Isolated T cell receptors and methods of use therefor

ABSTRACT

Provided are isolated TCRs, TCR-like molecules, and portions thereof that bind to phosphopeptide-HLA-A2 complexes. The isolated TCRs, TCR-like molecules, or portions are optionally soluble TCRs, TCR-like molecules, or portions. Also provided are isolated nucleic acids encoding the disclosed TCRs, TCR-like molecules, or portions; host cells that contain the disclosed TCRs, TCR-like molecules, or portions; pharmaceutical compositions that include the disclosed TCRs, TCR-like molecules, portions, nucleic acids, and/or T cells; kits; and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The presently disclosed subject matter claims the benefit of U.S.Provisional Patent Application Ser. No. 61/979,854, filed Apr. 15, 2014,the entire disclosure of which is incorporated herein by reference inits entirety.

GRANT STATEMENT

This invention was made with government support under grant numbersAI020963, CA134060, and CA044579 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing associated with the instant disclosure has beenelectronically submitted to the United States Patent and TrademarkOffice as a 146 kilobyte ASCII text file created on Apr. 2, 2015 andentitled “3062_9PCT_ST25.txt”. The Sequence Listing submitted viaEFS-Web is identical with respect to the sequences disclosure to theSequence Listing submitted to the United States Patent and TrademarkOffice on Apr. 15, 2014 as a 146 kilobyte ASCII text file created onMar. 18, 2014 and entitled “3062_9_ST25.txt”. Both Sequence Listings arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

The presently disclosed subject matter relates to the area ofdiagnostics and therapeutics. In particular, it relates toimmunotherapies and diagnostics in the context of proliferative diseasessuch as but not limited to cancer.

BACKGROUND

The mammalian immune system has evolved a variety of mechanisms toprotect the host from cancerous cells. An important component of thisresponse is mediated by cells referred to as T cells. Cytotoxic Tlymphocytes (CTL) are specialized T cells that primarily function byrecognizing and killing cancerous cells or infected cells, but they canalso function by secreting soluble molecules referred to as cytokinesthat can mediate a variety of effects on the immune system. T helpercells primarily function by recognizing antigen on specialized antigenpresenting cells, and in turn secreting cytokines that activate B cells,T cells, and macrophages. A variety of evidence suggests thatimmunotherapy designed to stimulate a tumor-specific CTL response wouldbe effective in controlling cancer. For example, it has been shown thathuman CTL recognize sarcomas (Slovin et al., 1986), renal cellcarcinomas (Schendel et al., 1993), colorectal carcinomas (Jacob et al.,1997), ovarian carcinomas (Peoples et al., 1993), pancreatic carcinomas(Peiper et al., 1997), squamous tumors of the head and neck (Yasumura etal., 1993), and squamous carcinomas of the lung (Slingluff et al., 1994;Yoshino et al., 1994). The largest number of reports of humantumor-reactive CTLs, however, has concerned melanomas (Boon et al.,1994). The ability of tumor-specific CTL to mediate tumor regression, inboth human (Parmiani et al., 2002; Weber, 2002) and animal models,suggests that methods directed at increasing CTL activity would likelyhave a beneficial effect with respect to tumor treatment.

Clinical trials using adoptive cellular therapy and active vaccinationhave demonstrated the importance of CD8 T-cells in controlling cancer(Morgan et al., 2006; Rosenberg, 2008; Hiugano et al., 2009;Schwartzentruber et al., 2011). A large number of tumor-associatedantigens (TAA) recognized by CD8 T-cells have been identified in thelast 20 years, and clinical tumor regressions have been associated withimmunotherapies based on some of them (Slingluff et al., 2004;Rosenberg, 2008). However, cancer vaccines targeting a range of TAA haveinduced disappointing clinical response rates of 3-6% (Rosenberg et al.,2004). The repertoire of TAA include: i) neoantigens formed by mutationsin cellular proteins; ii) antigens induced by oncogenic viruses; iii)cancer-testis antigens normally expressed only in germ-line cells; andiv) tissue-specific differentiation antigens (Williamson et al., 2006).Only a small number of TAA source proteins have been linked to eitherinitial cellular transformation processes or later tumorigenic processessuch as angiogenesis and metastasis (Hogan et al., 1998; Simpson et al.,2005). Targeting TAA derived from proteins that are vital for a cancercell's survival and metastatic potential is attractive, sincedown-regulation and/or mutation of genes encoding these proteins as ameans of immune evasion could compromise cellular malignancy (Dunn etal., 2004; Hirohashi et al., 2009).

As such, TCRs can be employed for various purposes for which howantibody molecules have been utilized. One challenge with respect toTCRs as opposed to antibodies, however, is that the former are notsecreted from the cells in which they are made. This can limit theutility of TCRs as therapeutic and/or diagnostic agents. Thesechallenges have been met to varying degrees of success by the productionof soluble TCRs.

Several methods for producing soluble TCRs and TCR-like molecules haverecent been reported. For example, U.S. Patent Application PublicationNo. 2008/0015139 of Lichterfeld et al. describes the production and useof soluble TCRs for the detection and treatment of viral infections. PCTInternational Patent Application Publication No. WO 2013/057586 ofWalseng et al. describes various additional methods for producingsoluble TCRs, such as isolation of α and β chains from bacterialinclusion bodies (see also Richman & Kranz, 2007) and STAR™ technology(Altor Bioscience Corporation, Miramar, Fla., United States of America),in which hybrid soluble TcR-Ig molecules are connected via a flexiblelinker (see also Mosquera et al., 2005).

Thus, soluble TCRs are useful as diagnostic and/or therapeutic tools.They can be employed to detect cells that express TAAs such as, but notlimited to peptides derived from TAAs complexed with MHC molecules.Additionally, soluble TCRs can be used to deliver a therapeutic agent,including but not limited to a cytotoxic compound or animmunostimulating compound, to cells presenting a particular TAA-derivedpeptide.

The interaction of a TCR with HLA-bound antigens including, but notlimited to a peptide derived from a TAA, results in cytotoxic Tlymphocytes (CTLs) killing cells that express the antigen (e.g., acancer cell) and/or secreting cytokines in response to a cancer cell.This process involves the interaction of the T cell receptor, located onthe surface of the CTL, with what is generically referred to as anMHC-peptide complex which is located on the surface of the cancerouscell. Major histocompatibility complex (MHC)-encoded molecules have beensubdivided into two types, and are referred to as class I and class IIMHC-encoded molecules. In the human immune system, MHC molecules arereferred to as human leukocyte antigens (HLA). Within the MHC complex,located on chromosome six, are three different loci that encode forclass I MHC molecules. MHC molecules encoded at these loci are referredto as HLA-A, HLA-B, and HLA-C. The genes that can be encoded at each ofthese loci are extremely polymorphic, and thus, different individualswithin the population express different class I MHC molecules on thesurface of their cells. HLA-A1, HLA-A2, HLA-A3, HLA-B7, HLA-B14,HLA-B27, and HLA-B44 are examples of different class I MHC moleculesthat can be expressed from these loci.

The peptides which associate with the MHC molecules can either bederived from proteins made within the cell, in which case they typicallyassociate with class I MHC molecules (Rock & Goldberg, 1999); or theycan be derived from proteins which are acquired from outside of thecell, in which case they typically associate with class II MHC molecules(Watts, 1997). The peptides that evoke a cancer-specific CTL responsemost typically associate with class I MHC molecules. The peptidesthemselves are typically nine amino acids in length, but can vary from aminimum length of eight amino acids to a maximum of fourteen amino acidsin length. Tumor antigens may also bind to class II MHC molecules onantigen presenting cells and provoke a T helper cell response. Thepeptides that bind to class II MHC molecules are generally twelve tonineteen amino acids in length, but can be as short as ten amino acidsand as long as thirty amino acids.

The process by which intact proteins are degraded into peptides isreferred to as antigen processing. Two major pathways of antigenprocessing occur within cells (Rock & Goldberg, 1999). One pathway,which is largely restricted to professional antigen presenting cellssuch as dendritic cells, macrophages, and B cells, degrades proteinsthat are typically phagocytosed or endocytosed into the cell. Peptidesderived from this pathway can be presented on either class I or to classII MHC molecules. A second pathway of antigen processing is present inessentially all cells of the body. This second pathway primarilydegrades proteins that are made within the cells, and the peptidesderived from this pathway primarily bind to class I MHC molecules.Antigen processing by this latter pathway involves polypeptide synthesisand proteolysis in the cytoplasm, followed by transport of peptides tothe plasma membrane for presentation. These peptides, initially beingtransported into the endoplasmic reticulum of the cell, becomeassociated with newly synthesized class I MHC molecules and theresulting complexes are then transported to the cell surface. Peptidesderived from membrane and secreted proteins have also been identified.In some cases these peptides correspond to the signal sequence of theproteins which is cleaved from the protein by the signal peptidase. Inother cases, it is thought that some fraction of the membrane andsecreted proteins are transported from the endoplasmic reticulum intothe cytoplasm where processing subsequently occurs. Once bound to theclass I MHC molecule, the peptides are recognized by antigen-specificreceptors on CTL. Several methods have been developed to identify thepeptides recognized by CTL, each method of which relies on the abilityof a CTL to recognize and kill only those cells expressing theappropriate class I MHC molecule with the peptide bound to it. Mereexpression of the class I MHC molecule is insufficient to trigger theCTL to kill the target cell if the antigenic peptide is not bound to theclass I MHC molecule. Such peptides can be derived from a non-selfsource, such as a pathogen (for example, following the infection of acell by a bacterium or a virus) or from a self-derived protein within acell, such as a cancerous cell. The tumor antigens from which thepeptides are derived can broadly be categorized as differentiationantigens, cancer/testis antigens, mutated gene products, widelyexpressed proteins, viral antigens and most recently, phosphopeptidesderived from dysregulated signal transduction pathways. (Zarling et al.,2006).

Immunization with cancer-derived, class I or class II MHC-encodedmolecule associated peptides, or with a precursor polypeptide or proteinthat contains the peptide, or with a gene that encodes a polypeptide orprotein containing the peptide, are forms of immunotherapy that can beemployed in the treatment of colorectal cancer. Identification of theimmunogens is a necessary first step in the formulation of theappropriate immunotherapeutic agent or agents. Although a large numberof tumor-associated peptide antigens recognized by tumor reactive CTLhave been identified, there are few examples of antigens that arederived from proteins that are selectively expressed on a broad array oftumors, as well as associated with cellular proliferation and/ortransformation.

Attractive candidates for this type of antigen are peptides derived fromproteins that are differentially phosphorylated on serine (Ser),threonine (Thr), and/or tyrosine (Tyr; Zarling et al., 2000). Due to theincreased and dysregulated phosphorylation of cellular proteins intransformed cells as compared to normal cells, tumors are likely topresent a unique subset of phosphorylated peptides on the cell surfacethat are available for recognition by cytotoxic T-lymphocytes (CTL).Presently, there is no way to predict which protein phosphorylationsites in a cell will be unique to tumors, survive the antigen processingpathway, and be presented to the immune system in the context of 8-14residue phosphopeptides bound to class I MHC molecules. However,thirty-six phosphopeptides were disclosed as presented in associationwith HLA-A*0201 on cancer cells (see Table 1 of Zarling et al., 2006).

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently disclosed subject matter providesisolated T cell receptors (TCRs), TCR-like molecules, and portionsthereof that bind to phosphopeptide/MHC complexes (optionally,phosphopeptide/HLA-A2 complexes). In some embodiments, thephosphopeptide is RVApSPTSGV (SEQ ID NO: 2). In some embodiments, theisolated TCR, TCR-like molecule, or portion thereof comprises an alphachain comprising a CDR3 region comprising AVSEGADRLT (amino acids111-120 of SEQ ID NO: 4) and a beta chain comprising a CDR3 regioncomprising ASSLLDSSYEQY (amino acids 112-123 of SEQ ID NO: 6), and insome embodiments the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising a CDR3 region comprising AVSAGSGGKLT(amino acids 111-122 of SEQ ID NO: 8) and a beta chain comprising a CDR3region comprising ASSDRDNYAEQF (amino acids 110-122 of SEQ ID NO: 10).In some embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a TRAV9D-4*04 V region, aTRAJ45*01 J region, and a TRAC*01 constant region, and a beta chaincomprising a TRBV14*01 V region, a TRBJ2-7*01 J region, a TRBD1*01 Dregion, and a TRBC2*03 constant region. In some embodiments, the alphachain comprises a TRAV9D-4*04 V region, a TRAJ44*01 J region, and aTRAC*01 constant region, and the beta chain comprises a TRBV13-3*01 Vregion, a TRBJ2-1*01 J region, a TRBD1*01 D region, and a TRBC2*03constant region. In some embodiments, the isolated TCR, TCR-likemolecule, or portion thereof comprises an alpha chain comprising anamino acid sequence at least 90% or 95% identical to SEQ ID NO: 4 and abeta chain comprising an amino acid sequence at least 90% or 95%identical to SEQ ID NO: 6; or an alpha chain comprising an amino acidsequence at least 90% or 95% identical to SEQ ID NO: 8 and a beta chaincomprising an amino acid sequence at least 90% or 95% identical to SEQID NO: 10. In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a CDR1 regioncomprising YSGTPY (amino acids 46-51 of SEQ ID NO: 4) and/or a CDR2region comprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 4), and abeta chain comprising a CDR1 region comprising SGHDT (amino acids 46-50of SEQ ID NO: 6) and/or a CDR2 region comprising FRDEAV (amino acids68-73 of SEQ ID NO: 6). In some embodiments, the alpha chain comprises aCDR1 region comprising YSGTPY (amino acids 46-50 of SEQ ID NO: 8) and/ora CDR2 region comprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 8),and the beta chain comprises a CDR1 region comprising NNHDY (amino acids45-49 of SEQ ID NO: 10) and/or a CDR2 region comprising SYVADS (aminoacids 67-72 of SEQ ID NO: 10). In some embodiments, the isolated TCR,TCR-like molecule, or portion thereof is a soluble TCR, TCR-likemolecule, or portion thereof comprising an alpha chain comprising a CDR1region comprising YSGTPY (amino acids 46-51 of SEQ ID NO: 4), a CDR2region comprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 4), and aCDR3 region comprising AVSEGADRLT (amino acids 111-120 of SEQ ID NO: 4);and a beta chain comprising a CDR1 region comprising SGHDT (amino acids46-50 of SEQ ID NO: 6), a CDR2 region comprising FRDEAV (amino acids68-73 of SEQ ID NO: 6), and a CDR3 region comprising ASSLLDSSYEQY (aminoacids 112-123 of SEQ ID NO: 6). In some embodiments, the alpha chaincomprises a CDR1 region comprising YSGTPY (amino acids 46-50 of SEQ IDNO: 8), a CDR2 region comprising YYSGDPVV (amino acids 69-76 of SEQ IDNO: 8), and a CDR3 region comprising AVSAGSGGKLT (amino acids 111-122 ofSEQ ID NO: 8); and the beta chain comprises a CDR1 region comprisingNNHDY (amino acids 45-49 of SEQ ID NO: 10), a CDR2 region comprisingSYVADS (amino acids 67-72 of SEQ ID NO: 10), and a CDR3 regioncomprising ASSDRDNYAEQF (amino acids 110-122 of SEQ ID NO: 10).

In some embodiments, the phosphopeptide is GLLGpSPVRA (SEQ ID NO: 12).In some embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a CDR3 region comprisingAVKPGGYKVV (amino acids 112-121 of SEQ ID NO: 14) and a beta chaincomprising a CDR3 region comprising ASGGDTQY (amino acids 121-129 of SEQID NO: 16). In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a TRAV3-3*02 Vregion, a TRAJ12*01 J region, and a TRAC*01 constant region, and a betachain comprising a TRBV13-2*01 V region, a TRBJ2-5*01 J region, aTRBD2*01 D region, and a TRBC2*03 constant region. In some embodiments,the isolated TCR, TCR-like molecule, or portion thereof comprises analpha chain comprising an amino acid sequence at least 90% or 95%identical to SEQ ID NO: 14 and a beta chain comprising an amino acidsequence at least 90% or 95% identical to SEQ ID NO: 16. In someembodiments, the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising a CDR1 region comprising DPNSYY(amino acids 48-53 of SEQ ID NO: 14) and/or a CDR2 region comprisingVFSSTEI (amino acids 71-77 of SEQ ID NO: 14), and a beta chaincomprising a CDR1 region comprising NNHNN (amino acids 56-60 of SEQ IDNO: 16) and/or a CDR2 region comprising SYGAGS (amino acids 78-83 of SEQID NO: 16). In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof is a soluble TCR, TCR-like molecule, or portion thereofcomprising an alpha chain comprising a CDR1 region comprising DPNSYY(amino acids 48-53 of SEQ ID NO: 14), a CDR2 region comprising VFSSTEI(amino acids 71-77 of SEQ ID NO: 14), and a CDR3 region comprisingAVKPGGYKVV (amino acids 112-121 of SEQ ID NO: 14); and a beta chaincomprising a CDR1 region comprising NNHNN (amino acids 56-60 of SEQ IDNO: 16), a CDR2 region comprising SYGAGS (amino acids 78-83 of SEQ IDNO: 16), and a CDR3 region comprising ASGGDTQY (amino acids 121-129 ofSEQ ID NO: 16).

In some embodiments, the phosphopeptide is RTFpSPTYGL (SEQ ID NO: 19).In some embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a CDR3 region comprisingVLSYSNNRIF (amino acids 111-120 of SEQ ID NO: 21) and a beta chaincomprising a CDR3 region comprising ASSLGGGEVF (amino acids 121-130 ofSEQ ID NO: 23), and in some embodiments the isolated TCR, TCR-likemolecule, or portion thereof comprises an alpha chain comprising a CDR3region comprising VLRYGGNNKLT (amino acids 111-121 of SEQ ID NO: 25) andbeta chain comprising a CDR3 region comprising ASRYRDTQY (amino acids110-118 of SEQ ID NO: 27). In some embodiments, the isolated TCR,TCR-like molecule, or portion thereof comprises an alpha chaincomprising a TRAV9D-4*02 V region, a TRAJ31*01 J region, and a TRAC*01constant region, and a beta chain comprising a TRBV12-1*01 V region, aTRBJ1-1*01/J1-1*02 J region, a TRBD1*01 D region, and a TRBC1*01constant region. In some embodiments, the alpha chain comprises aTRAV9D-4*02 V region, a TRAJ56*01 J region, and a TRAC*01 constantregion, and the beta chain comprises a TRBV13-3*01 V region, aTRBJ2-5*01 J region, a TRBD1*01 D region, and a TRBC2*03 constantregion. In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising an amino acidsequence at least 90% or 95% identical to SEQ ID NO: 21 and a beta chaincomprising an amino acid sequence at least 90% or 95% identical to SEQID NO: 23; or an alpha chain comprising an amino acid sequence at least90% or 95% identical to SEQ ID NO: 25 and a beta chain comprising anamino acid sequence at least 90% or 95% identical to SEQ ID NO: 27. Insome embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a CDR1 region comprisingYSGTPY (amino acids 46-51 of SEQ ID NO: 21) and/or a CDR2 regioncomprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 21), and a betachain comprising a CDR1 region comprising SGHSN (amino acids 56-60 ofSEQ ID NO: 23) and/or a CDR2 region comprising HYEKVE (amino acids 78-83of SEQ ID NO: 23). In some embodiments, the alpha chain comprises a CDR1region comprising YSGTPY (amino acids 46-51 of SEQ ID NO: 25) and/or aCDR2 region comprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 25),and the beta chain comprises a CDR1 region comprising NNHDY (amino acids45-49 of SEQ ID NO: 27) and/or a CDR2 region comprising SYVADS (aminoacids 67-72 of SEQ ID NO: 27). In some embodiments, the isolated TCR,TCR-like molecule, or portion thereof is a soluble TCR, TCR-likemolecule, or portion thereof comprising alpha chain comprising a CDR1region comprising YSGTPY (amino acids 46-51 of SEQ ID NO: 21), a CDR2region comprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 21), and aCDR3 region comprising VLSYSNNRIF (amino acids 111-120 of SEQ ID NO:21); and a beta chain comprising a CDR1 region comprising SGHSN (aminoacids 56-60 of SEQ ID NO: 23), a CDR2 region comprising HYEKVE (aminoacids 78-83 of SEQ ID NO: 23), and a CDR3 region comprising ASSLGGGEVF(amino acids 121-130 of SEQ ID NO: 23). In some embodiments, the alphachain comprises a CDR1 region comprising YSGTPY (amino acids 46-51 ofSEQ ID NO: 25), a CDR2 region comprising YYSGDPVV (amino acids 69-76 ofSEQ ID NO: 25), and a CDR3 region comprising VLRYGGNNKLT (amino acids111-121 of SEQ ID NO: 25); and the beta chain comprises a CDR1 regioncomprising NNHDY (amino acids 45-49 of SEQ ID NO: 27), a CDR2 regioncomprising SYVADS (amino acids 67-72 of SEQ ID NO: 27), and a CDR3region comprising ASRYRDTQY (amino acids 110-118 of SEQ ID NO: 27).

In some embodiments, the phosphopeptide is YLDpSGIHSGV (SEQ ID NO: 29).In some embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a CDR3 region comprisingAIPPGTGSKLS (amino acids 108-118 of SEQ ID NO: 32) and a beta chaincomprising a CDR3 region comprising ASSQGQKGY (amino acids 110-118 ofSEQ ID NO: 34). In some embodiments, the isolated TCR, TCR-likemolecule, or portion thereof comprises an alpha chain comprising aTRAV13*02 V region, a TRAJ58*01 J region, and a TRAC*01 constant region,and a beta chain comprising a TRBV5*01 V region, a TRBJ2-7*01 J region,a TRBD1*01 D region, and a TRBC2*03 constant region. In someembodiments, the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising an amino acid sequence at least 90%or 95% identical to SEQ ID NO: 32 and a beta chain comprising an aminoacid sequence at least 90% or 95% identical to SEQ ID NO: 34. In someembodiments, the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising a CDR1 region comprising STATR(amino acids 47-51 of SEQ ID NO: 32) and/or a CDR2 region comprisingNPSGT (amino acids 69-73 of SEQ ID NO: 32), and a beta chain comprisinga CDR1 region comprising LGHNA (amino acids 45-49 of SEQ ID NO: 34)and/or a CDR2 region comprising YNLKQL (amino acids 67-72 of SEQ ID NO:34). In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof is a soluble TCR, TCR-like molecule, or portion thereofcomprising an alpha chain comprising a CDR1 region comprising STATR(amino acids 47-51 of SEQ ID NO: 32), a CDR2 region comprising NPSGT(amino acids 69-73 of SEQ ID NO: 32), and a CDR3 region comprisingAIPPGTGSKLS (amino acids 108-118 of SEQ ID NO: 32); and a beta chaincomprising a CDR1 region comprising LGHNA (amino acids 45-49 of SEQ IDNO: 34), a CDR2 region comprising YNLKQL (amino acids 67-72 of SEQ IDNO: 34), and a CDR3 region comprising ASSQGQKGY (amino acids 110-118 ofSEQ ID NO: 34).

In some embodiments, the phosphopeptide is YLDpSGIHSGA (SEQ ID NO: 30).In some embodiments, the isolated TCR, TCR-like molecule, or portionthereof comprises an alpha chain comprising a CDR3 region comprisingATGPNTNKVV (amino acids 110-119 of SEQ ID NO: 36) and a beta chaincomprising a CDR3 region comprising ASSQGGAEQF (amino acids 110-119 ofSEQ ID NO: 38). In some embodiments, the isolated TCR, TCR-likemolecule, or portion thereof comprises an alpha chain comprising aTRAV8D-2*02 V region, a TRAJ34*02 J region, and a TRAC*01 constantregion, and a beta chain comprising a TRBV5*01 V region, a TRBJ2-1*01 Jregion, a TRBD2*01 D region, and a TRBC2*03 constant region. In someembodiments, the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising an amino acid sequence at least 90%or 95% identical to SEQ ID NO: 36 and a beta chain comprising an aminoacid sequence at least 90% or 95% identical to SEQ ID NO: 38. In someembodiments, the isolated TCR, TCR-like molecule, or portion thereofcomprises an alpha chain comprising a CDR1 region comprising TYTTV(amino acids 47-51 of SEQ ID NO: 36) and/or a CDR2 region comprisingIRSNERE (amino acids 69-75 of SEQ ID NO: 36), and a beta chaincomprising a CDR1 region comprising LGHKA (amino acids 45-49 of SEQ IDNO: 38) and/or a CDR2 region comprising YNLKQL (amino acids 67-72 of SEQID NO: 38). In some embodiments, the isolated TCR, TCR-like molecule, orportion thereof is a soluble TCR, TCR-like molecule, or portion thereofcomprising an alpha chain comprising a CDR1 region comprising TYTTV(amino acids 47-51 of SEQ ID NO: 36), a CDR2 region comprising IRSNERE(amino acids 69-75 of SEQ ID NO: 36), and a CDR3 region comprisingATGPNTNKVV (amino acids 110-119 of SEQ ID NO: 36); and a beta chaincomprising a CDR1 region comprising LGHKA (amino acids 45-49 of SEQ IDNO: 38), a CDR2 region comprising YNLKQL (amino acids 67-72 of SEQ IDNO: 38), and a CDR3 region comprising ASSQGGAEQF (amino acids 110-119 ofSEQ ID NO: 38).

In some embodiments, the isolated and/or soluble TCR, TCR-like molecule,or portion thereof is conjugated to an active agent. In someembodiments, the active agent is selected from the group consisting of adetectable label, an immunostimulatory molecule, and a therapeuticagent. In some embodiments, the detectable label is selected from thegroup consisting of biotin, streptavidin, an enzyme or catalyticallyactive fragment thereof, a radionuclide, a nanoparticle, a paramagneticmetal ion, or a fluorescent, phosphorescent, or chemiluminescentmolecule. In some embodiments, the immunostimulatory molecule is a CD3agonist, optionally an anti-CD3 antibody. In some embodiments, thetherapeutic agent is selected from the group consisting of an alkylatingagent, an antimetabolite, a natural product having pharmacologicalactivity, a mitotic inhibitor, an antibiotic, a cytotoxic agent, and achemotherapeutic agent.

In some embodiments, the isolated and/or soluble TCR, TCR-like molecule,or portion thereof is humanized, comprises a human constant domain, orboth.

The presently disclosed subject matter also provides isolated nucleicacids encoding the isolated and/or soluble TCRs, TCR-like molecules, orportions thereof disclosed herein. In some embodiments, the nucleicacids are present in vectors, optionally expression vectors. In someembodiments, the nucleic acids are present in expression vectors undertranscriptional and optionally translational control of regulatorysequences sufficient to express the nucleic acids in cells, optionallyprokaryotic cells and optionally eukaryotic cells. In some embodiments,the cells are mammalian cells, and in some embodiments the mammaliancells are human cells. In some embodiments, an isolated nucleic acid ofthe presently disclosed subject matter is a complementary DNA (cDNA)encoding any one of SEQ ID NOs: 4, 6, 8, 10, 14, 16, 21, 23, 25, 27, 32,34, 36, or 38, or an extracellular portion thereof, optionally comprisesany of the CDR1/CDR2/CDR3 combinations disclosed herein. In someembodiments, when a cDNA of the presently disclosed subject matterencodes both an alpha chain and a beta chain, the cDNA includes aninternal ribosome entry site (IRES) such that translation of the cDNA ina host cell produces both the alpha chain and the beta chain encodedthereby.

The presently disclosed subject matter also provides host cells. In someembodiments, a host cell of the presently disclosed subject mattercomprises an isolated and/or soluble TCR, TCR-like molecule, or portionthereof as disclosed herein, an isolated nucleic acid as disclosedherein, or both.

The presently disclosed subject matter also provides isolated T cellscomprising one or more isolated and/or soluble TCRs, TCR-like molecules,and/or portions thereof disclosed herein, one or more isolated nucleicacids disclosed herein, or any combination thereof.

The presently disclosed subject matter also provides pharmaceuticalcompositions. In some embodiments, a pharmaceutical composition of thepresently disclosed subject matter comprises an isolated and/or solubleTCR, TCR-like molecule, or portion thereof as disclosed herein; anisolated nucleic acid as disclosed herein; an isolated T cell asdisclosed herein; or any combination thereof. In some embodiments,administration of a therapeutically effective amount of a pharmaceuticalcomposition as disclosed herein to a patient who has a tumor and/or acancer is capable of increasing the 5-year survival rate of the patientby at least 20 percent relative to average 5-year survival rates thatcould have been expected without treatment with the pharmaceuticalcomposition. In some embodiments, administration of a therapeuticallyeffective amount of a pharmaceutical composition as disclosed herein toa patient who has a tumor and/or a cancer is capable of increasing thesurvival rate of the patient by at least 20 percent relative to asurvival rate that could have been expected without treatment with thepharmaceutical composition. In some embodiments, administration of atherapeutically effective amount of a pharmaceutical composition asdisclosed herein to a patient who has a tumor and/or a cancer is capableof increasing the treatment response rate of the patient to the tumorand/or the cancer by at least 20 percent relative to a treatment ratethat could have been expected without treatment with the pharmaceuticalcomposition. In some embodiments, administration of a therapeuticallyeffective amount of a pharmaceutical composition as disclosed herein toa patient who has a tumor and/or a cancer is capable of increasing theoverall median survival of the patient by at least two months relativeto an overall median survival that could have been expected withouttreatment with the pharmaceutical composition. In some embodiments, thepharmaceutical composition further comprises at least one peptidederived from MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2,MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE),SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL,H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP andTPS. In some embodiments, the pharmaceutical composition furthercomprises an adjuvant selected from the group consisting of montanideISA-51 (Seppic, Inc.), QS-21 (Aquila Pharmaceuticals, Inc), tetanushelper peptides, GM-CSF, cyclophosamide, bacillus Calmette-Guérin (BCG),corynbacterium parvum, levamisole, azimezone, isoprinisone,dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freund'sadjuvant (complete and incomplete), mineral gels, aluminum hydroxide(Alum), lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, dinitrophenol, diphtheria toxin (DT).

The presently disclosed subject matter also provides methods foradoptive T cell therapy. In some embodiments, the methods compriseadministering to a subject in need thereof an isolated T cell asdisclosed herein and/or a pharmaceutical composition as disclosedherein. In some embodiments, the presently disclosed methods furthercomprise administering to the subject an effective amount of CD4+ Thelper cells before, after, or concomitantly with the isolated T celland/or the pharmaceutical composition. In some embodiments, thepresently disclosed methods further comprise exposing the subject to atreatment that creates a lymphopenic environment in the subject, therebyenhancing engraftment and/or expansion of the isolated T cell. In someembodiments of the presently disclosed methods, the subject has a tumorand/or a cancer, optionally a tumor and/or a cancer selected from thegroup consisting of pancreatic cancer, hepatocellular carcinoma,neuroblastoma, breast cancer, glioblastoma, and colorectal cancer, andthe administered isolated T cell and/or a component of the administeredpharmaceutical composition specifically binds to a tumor-associatedantigen expressed by cells of the tumor and/or the cancer.

The presently disclosed subject matter also provides methods fortreating tumors and/or cancers in patients, wherein said tumors and/orcancers bear one or more immunologically reactive tumor-specificantigens. In some embodiments, the methods comprise transformingperipheral blood lymphocytes isolated from a patient, optionally Tcells, further optionally cytotoxic CD8+ T cells, with an expressionvector, wherein the expression vector comprises a nucleotide sequenceencoding an isolated and/or soluble TCR, TCR-like molecule, or portionthereof as disclosed herein; and administering the transformed cells tothe patient, the transformed cells being targeted to the tumor, therebytreating the tumor.

The presently disclosed subject matter also provides methods fordirecting the immune response of a patient toward a predefined targetantigen. In some embodiments, the methods comprise transfecting alymphocyte with a recombinant DNA encoding an isolated and/or solubleTCR, TCR-like molecule, or portion thereof as disclosed herein; andadministering the transfected lymphocyte to the patient. In someembodiments, the patient is given a pre-treatment that partially orcompletely destroys or otherwise inactivates the patient's T cellcompartment. In some embodiments, the amount of transfected lymphocytesadministered to the patient is sufficient to engraft in the patient andoptionally partially or completely reconstitute the T cell compartmentof the patient. In some embodiments, the patient has a tumor and/or acancer, optionally a tumor and/or a cancer selected from the groupconsisting of pancreatic cancer, hepatocellular carcinoma,neuroblastoma, breast cancer, glioblastoma, and colorectal cancer. Insome embodiments, the predefined target antigen comprises aphosphopeptide comprising an amino acid sequence selected from the groupconsisting of RVApSPTSGV (SEQ ID NO: 2), GLLGpSPVRA (SEQ ID NO: 12),RTFpSPTYGL (SEQ ID NO: 19), YLDpSGIHSGV (SEQ ID NO: 29), and YLDpSGIHSGA(SEQ ID NO: 30).

In some embodiments, the presently disclosed subject matter alsoprovides methods for generating antigen-specific T cells. In someembodiments, the methods comprise providing a nucleic acid encoding anisolated and/or soluble TCR, TCR-like molecule, or portion thereof asdisclosed herein; introducing the nucleic acid into a T cell; andoptionally selecting a T cell that expresses the TCR, the TCR-likemolecule, or the portion thereof. In some embodiments, the presentlydisclosed methods further comprise identifying an antigen-specific Tcell that recognizes a complex of an MHC molecule and a phosphopeptide,wherein the phosphopeptide comprises an amino acid sequence selectedfrom the group consisting of RVApSPTSGV (SEQ ID NO: 2), GLLGpSPVRA (SEQID NO: 12), RTFpSPTYGL (SEQ ID NO: 19), YLDpSGIHSGV (SEQ ID NO: 29), andYLDpSGIHSGA (SEQ ID NO: 30).

The presently disclosed subject matter also provides in some embodimentsin vitro populations of T cells transfected with a nucleic acid (e.g.,mRNA, cDNA, or genomic DNA) encoding an isolated and/or soluble TCR,TCR-like molecule, or portion thereof of the presently disclosed subjectmatter.

The presently disclosed subject matter also provides methods fortreating and/or preventing cancer, said methods comprising administeringto a patient in need thereof a dose of a pharmaceutical composition asdisclosed herein.

The presently disclosed subject matter also provides in some embodimentsmethods for treating and/or preventing cancer comprising administeringto a patient in need thereof one or more doses of the isolated T cellsdisclosed herein, optionally isolated CD8+ T cells, wherein theadministered T cells are administered in combination with apharmaceutically acceptable carrier.

The presently disclosed subject matter also provides in some embodimentsmethods for making cancer vaccines. In some embodiments, the presentlydisclosed methods comprise combining a composition comprising aplurality of the isolated T cells of the presently disclosed subjectmatter with an adjuvant and/or a pharmaceutically acceptable carrier andplacing the composition comprising the plurality of isolated T cells andthe adjuvant and/or the pharmaceutical carrier into a syringe.

The presently disclosed subject matter also provides in some embodimentskits. In some embodiments, the kits comprise a cytokine and/or anadjuvant; and at least one composition comprising an isolated and/orsoluble T cell comprising a TCR, TCR-like molecule, or portion thereofas disclosed herein, an isolated T cell as disclosed herein, apharmaceutical composition as disclosed herein, or any combinationthereof. In some embodiments, a presently disclosed kit comprises aplurality of T cells that together comprise at least 1, 2, 3, 4, 5, 6,7, or more different isolated and/or soluble TCRs, TCR-like molecules,or portions thereof as disclosed herein. In some embodiments, thecytokine is selected from the group consisting of transforming growthfactors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I andinsulin-like growth factor-II; erythropoietin (EPO); osteoinductivefactors; interferons such as IFNα, IFNβ, and IFNγ; colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). Insome embodiments, the cytokine is selected from the group consisting ofnerve growth factors such as NGF-β; platelet-growth factor; transforminggrowth factors (TGFs) such as TGF-α and TGF-β; insulin-like growthfactor-I and insulin-like growth factor-II; erythropoietin (EPO);osteoinductive factors; interferons such as IFNα, IFNβ, and IFNγ; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT.In some embodiments, the adjuvant is selected from the group consistingof montanide ISA-51 (Seppic, Inc), QS-21 (Aquila Pharmaceuticals, Inc),tetanus helper peptides, GM-CSF, cyclophosamide, bacillusCalmette-Guérin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freund's adjuvant (complete and incomplete), mineral gels,aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, dinitrophenol, diphtheria toxin (DT). In someembodiments, the presently disclosed kit further comprises at least onepeptide derived from MelanA (MART-I), gp100 (Pmel 17), tyrosinase,TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE,NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP andTPS. In some embodiments, the at least one target peptide comprises anamino acid sequence selected from among SEQ ID NOs: 2, 12, 19, 29, and30, or an antigenic portion thereof.

The presently disclosed subject matter also provides in vitropopulations of CD8+ T cells capable of being activated upon beingbrought into contact with a population of dendritic cells. In someembodiments, the in vitro population of CD8+ T cells comprise one ormore TCRs that bind to a complex of an HLA molecule and a phosphopeptideselected from the group consisting of RVApSPTSGV (SEQ ID NO: 2),GLLGpSPVRA (SEQ ID NO: 12), RTFpSPTYGL (SEQ ID NO: 19), YLDpSGIHSGV (SEQID NO: 29), and YLDpSGIHSGA (SEQ ID NO: 30).

In some embodiments, the presently disclosed subject matter alsoprovides methods for detecting the presence of a phosphopeptide in abiological sample suspected of containing the phosphopeptide. In someembodiments, the methods comprise contacting the biological sample witha TCR, TCR-like molecule, or portion thereof of the presently disclosedsubject matter (optionally a detectably labeled TCR, TCR-like molecule,or portion thereof); and detecting the TCR, TCR-like molecule, orportion thereof either directly or indirectly. In some embodiments, theTCR, TCR-like molecule, or portion thereof comprises an alpha chaincomprising a CDR3 region comprising AVSEGADRLT (amino acids 111-120 ofSEQ ID NO: 4) and a beta chain comprising a CDR3 region comprisingASSLLDSSYEQY (amino acids 112-123 of SEQ ID NO: 6); an alpha chaincomprising a CDR3 region comprising AVSAGSGGKLT (amino acids 111-122 ofSEQ ID NO: 8); and a beta chain comprising a CDR3 region comprisingASSDRDNYAEQF (amino acids 110-122 of SEQ ID NO: 10); an alpha chaincomprising a CDR3 region comprising AVKPGGYKVV (amino acids 112-121 ofSEQ ID NO: 14) and a beta chain comprising a CDR3 region comprisingASGGDTQY (amino acids 121-129 of SEQ ID NO: 16); an alpha chaincomprising a CDR3 region comprising VLSYSNNRIF (amino acids 111-120 ofSEQ ID NO: 21) and a beta chain comprising a CDR3 region comprisingASSLGGGEVF (amino acids 121-130 of SEQ ID NO: 23); an alpha chaincomprising a CDR3 region comprising VLRYGGNNKLT (amino acids 111-121 ofSEQ ID NO: 25) and beta chain comprising a CDR3 region comprisingASRYRDTQY (amino acids 110-118 of SEQ ID NO: 27); an alpha chaincomprising a CDR3 region comprising AIPPGTGSKLS (amino acids 108-118 ofSEQ ID NO: 32) and a beta chain comprising a CDR3 region comprisingASSQGQKGY (amino acids 110-118 of SEQ ID NO: 34); or an alpha chaincomprising a CDR3 region comprising ATGPNTNKVV (amino acids 110-119 ofSEQ ID NO: 36) and a beta chain comprising a CDR3 region comprisingASSQGGAEQF (amino acids 110-119 of SEQ ID NO: 38). In some embodiments,the biological sample is a patient biopsy and the detecting step isdiagnostic of the presence of tumor cells and/or cancer cells in thepatient biopsy.

In some embodiments, the presently disclosed subject matter alsoprovides methods for diagnosing a tumor and/or a cancer in a subject. Insome embodiments, the methods comprise contacting a biological sampleisolated from the subject with a TCR, TCR-like molecule, or portionthereof of the presently disclosed subject matter, optionally adetectably labeled TCR, TCR-like molecule, or portion thereof, anddetecting the TCR, TCR-like molecule, or portion thereof bound to thebiological sample directly or indirectly, wherein detecting the TCR,TCR-like molecule, or portion thereof bound to the biological sample isindicative of a tumor and/or a cancer in the subject. In someembodiments, the TCR, TCR-like molecule, or portion thereof comprises analpha chain comprising a CDR1 region comprising YSGTPY (amino acids46-51 of SEQ ID NO: 4), a CDR2 region comprising YYSGDPVV (amino acids69-76 of SEQ ID NO: 4), a CDR3 region comprising AVSEGADRLT (amino acids111-120 of SEQ ID NO: 4), or any combination thereof; and a beta chaincomprising a CDR1 region comprising SGHDT (amino acids 46-50 of SEQ IDNO: 6), a CDR2 region comprising FRDEAV (amino acids 68-73 of SEQ ID NO:6), a CDR3 region comprising ASSLLDSSYEQY (amino acids 112-123 of SEQ IDNO: 6), or any combination thereof. In some embodiments, the TCR,TCR-like molecule, or portion thereof comprises an alpha chaincomprising a CDR1 region comprising YSGTPY (amino acids 46-50 of SEQ IDNO: 8), a CDR2 region comprising YYSGDPVV (amino acids 69-76 of SEQ IDNO: 8), a CDR3 region comprising AVSAGSGGKLT (amino acids 111-122 of SEQID NO: 8), or any combination thereof; and a beta chain comprising aCDR1 region comprising NNHDY (amino acids 45-49 of SEQ ID NO: 10), aCDR2 region comprising SYVADS (amino acids 67-72 of SEQ ID NO: 10), aCDR3 region comprising ASSDRDNYAEQF (amino acids 110-122 of SEQ ID NO:10), or any combination thereof. In some embodiments, the TCR, TCR-likemolecule, or portion thereof comprises an alpha chain comprising a CDR1region comprising DPNSYY (amino acids 48-53 of SEQ ID NO: 14), a CDR2region comprising VFSSTEI (amino acids 71-77 of SEQ ID NO: 14), a CDR3region comprising AVKPGGYKVV (amino acids 112-121 of SEQ ID NO: 14), orany combination thereof; and a beta chain comprising a CDR1 regioncomprising NNHNN (amino acids 56-60 of SEQ ID NO: 16), a CDR2 regioncomprising SYGAGS (amino acids 78-83 of SEQ ID NO: 16), a CDR3 regioncomprising ASGGDTQY (amino acids 121-129 of SEQ ID NO: 16), or anycombination thereof. In some embodiments, the TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a CDR1 regioncomprising YSGTPY (amino acids 46-51 of SEQ ID NO: 21), a CDR2 regioncomprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 21), a CDR3 regioncomprising VLSYSNNRIF (amino acids 111-120 of SEQ ID NO: 21), or anycombination thereof; and a beta chain comprising a CDR1 regioncomprising SGHSN (amino acids 56-60 of SEQ ID NO: 23), a CDR2 regioncomprising HYEKVE (amino acids 78-83 of SEQ ID NO: 23), a CDR3 regioncomprising ASSLGGGEVF (amino acids 121-130 of SEQ ID NO: 23), or anycombination thereof. In some embodiments, the TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a CDR1 regioncomprising YSGTPY (amino acids 46-51 of SEQ ID NO: 25), a CDR2 regioncomprising YYSGDPVV (amino acids 69-76 of SEQ ID NO: 25), a CDR3 regioncomprising VLRYGGNNKLT (amino acids 111-121 of SEQ ID NO: 25), or anycombination thereof; and a beta chain comprising a CDR1 regioncomprising NNHDY (amino acids 45-49 of SEQ ID NO: 27), a CDR2 regioncomprising SYVADS (amino acids 67-72 of SEQ ID NO: 27), a CDR3 regioncomprising ASRYRDTQY (amino acids 110-118 of SEQ ID NO: 27), or anycombination thereof. In some embodiments, the TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a CDR1 regioncomprising STATR (amino acids 47-51 of SEQ ID NO: 32), a CDR2 regioncomprising NPSGT (amino acids 69-73 of SEQ ID NO: 32), a CDR3 regioncomprising AIPPGTGSKLS (amino acids 108-118 of SEQ ID NO: 32), or anycombination thereof; and a beta chain comprising a CDR1 regioncomprising LGHNA (amino acids 45-49 of SEQ ID NO: 34), a CDR2 regioncomprising YNLKQL (amino acids 67-72 of SEQ ID NO: 34), a CDR3 regioncomprising ASSQGQKGY (amino acids 110-118 of SEQ ID NO: 34), or anycombination thereof. In some embodiments, the TCR, TCR-like molecule, orportion thereof comprises an alpha chain comprising a CDR1 regioncomprising TYTTV (amino acids 47-51 of SEQ ID NO: 36), a CDR2 regioncomprising IRSNERE (amino acids 69-75 of SEQ ID NO: 36), a CDR3 regioncomprising ATGPNTNKVV (amino acids 110-119 of SEQ ID NO: 36), or anycombination thereof; and a beta chain comprising a CDR1 regioncomprising LGHKA (amino acids 45-49 of SEQ ID NO: 38), a CDR2 regioncomprising YNLKQL (amino acids 67-72 of SEQ ID NO: 38), a CDR3 regioncomprising ASSQGGAEQF (amino acids 110-119 of SEQ ID NO: 38), or anycombination thereof.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the presently disclosed subject mattercan be obtained by reference to the accompanying Figures, whenconsidered in conjunction with the subsequent Detailed Description. Theembodiments illustrated in the Figures are intended to be exemplaryonly, and should not be construed as limiting the presently disclosedsubject matter to the illustrated embodiments.

FIGS. 1A-1D are a series of graphs showing that phosphopeptides fromIRS-2 and CDC25b are immunogenic in vitro for human CD8 T-cells and invivo for AAD transgenic mice. Bulk (FIGS. 1A and 1B) and memory(CD45RO⁺; FIG. 1C) CD8 T-cells from HLA-A2⁺ donors were restimulated invitro in 6-12 replicate microcultures with pIRS-2₁₀₉₇₋₁₁₀₅₉,pCDC25b₃₈₋₄₆, M1₅₈₋₆₆ Flu, or Yellow Fever NS4B₂₁₄₋₂₂₂ peptide-pulsed DCfor 7 days. Antigen-specific T-cells were detected by ELISpot using T2stimulators pulsed with the indicated peptide. “p” refers to thephosphorylated form. In FIGS. 1A and 1B, each data point represents anindividual 7-day microculture. All donor responses were tested in threeseparate experiments with one representative experiment shown. FIG. 1Cis a bar graph showing the mean number of IFN-γ⁺ memory CD8 T-cells fromfour separate donor microcultures. Response to specific peptide isshown. FIG. 1D is a series of plots showing IFN-γ production by murineCD8 T-cell lines specific for indicated phosphopeptide followingco-culture with C1R-AAD or C1R-A2 targets pulsed with eitherpIRS-2₁₀₉₇₋₁₁₀₅ (left panel) or pCDC25b₃₈₋₄₆ (right panel) for 24 hours.C1R-AAD targets pulsed with the unphosphorylated peptides are indicatedwith triangles. Data representative of 3-4 separate experiments. FIG.1A: diamond—pIRS-2; Square—pCDC25b, triangle—yellow fever. FIG. 1C:Solid white boxes—donor 43; left to right hatching donor 44; right toleft tight hatching—donor 54; right to left broad hatching—donor 62.FIG. 1D: solid squares (left panel) or solid diamonds (rightpanel)—transfectants of the B lymphoblastoid cell line C1R expressing achimeric MHC class I molecule consisting of α1 and α2 domains of HLA-A2and α3 domain of H-2D^(d) (C1R-AAD); open squares (left panel) or opendiamonds (right panel)—transfectants of the B lymphoblastoid cell lineC1R expressing HLA-A2 (C1R-A2).

FIGS. 2A-2C are a series of plots showing that electroporation of invitro-transcribed (IVT) RNA encoding murine TCR chains resulted infunctional cell surface expression of phosphopeptide-specific TCR.Detection of murine TCR on the surface of TCR-deficient SupT1 (FIG. 2A)or human T lymphocytes (FIGS. 2A-2C) following electroporation of IVTRNA encoding the pIRS-2₁₀₉₇₋₁₁₀₅ TCR alpha and beta chains of SEQ IDNOs: 4 and 6. Note that expression of TCR chains resulted in cellsurface expression of CD3 on SupT1 cells. FIGS. 2B and 2C show stainingof mouse TCR on gated human CD8+ T cells. The data presented arerepresentative of eight separate experiments. FIG. 2B: - hour timepoint; -----: 24 hour time point;

48 hour time point;

72 hour time point;

: 72 hour time point, no RNA. FIG. 2C:

: FMO control; - - - : 5 days post-electroporation; -: 3 dayspost-electroporation.

FIGS. 3A-3C depict the results of experiments showing expression ofphosphopeptide-specific murine TCR in human CD8 T-cells conferredrecognition of HLA-A2⁺ targets and effector function. Human CD8 T-cellswere electroporated with IVT RNA encoding phosphopeptide-specific murineTCR αβ chains, and assayed 12-14 hours later. FIGS. 3A and 3B: leftpanels, Surface CD107a and/or intracellular IFN-γ were detected by flowcytometry on pIRS-2-specific (FIG. 3A) or pCDC25b-specific (FIG. 3B)human CD8 T-cells following co-culture with indicated C1R-AAD and C1R-A2unpulsed or peptide-pulsed targets. Right panels, In vitro cytotoxicityassay of pIRS-2-specific (FIG. 3A) or pCDC25b-specific (FIG. 3B) CD8T-cells was performed using phosphopeptide-pulsed (CFSE^(hi)) orunpulsed (CFSE^(lo)) C1R-A2 targets. For FIGS. 3A and 3B (left panels):x: no stimulators; open triangles: C1RA2 unpulsed; solid triangles:C1RA2+ pIRS-2; upside down open triangles: C1RA2+ IRS-2; open squares:C1RAAD unpulsed; solid squares: C1RAAD+pIRS-2. For FIGS. 3A and 3B(right panels): hatched boxes—effector (E) to target (T) ration 3:1;open boxes—E:T ratio 1:1. FIG. 3C is a series of plots showing surfaceCD107a and intracellular IFN-γ detected on pIRS-2-specific orpCDC25b-specific human CD8 T-cells following co-culture with theindicated human cancer cells (i.e., Mel Swift, 1102Mel, SK-Mel-28, andOV-90). No RNA controls underwent electroporation with no addition ofIVT RNA. Antigen expression was determined by Western blot and is shownin FIGS. 4 and 5. For all panels, data are representative of duplicate(triplicate for In vitro cytotoxicity assay) determinations in 2-5experiments.

FIGS. 4A-4D depict the results of experiments showing thatpIRS-2₁₀₉₇₋₁₁₀₅ was endogenously processed and presented by cancer cellsof multiple histological types. FIG. 4A is a series of immunoblotsshowing expression of pSer¹¹⁰⁰-IRS-2 (top), total IRS-2 (middle), andGAPDH (bottom) in extracts representing 1.5×10⁵ cell equivalents of theindicated cell lines. pSer¹¹⁰⁰-IRS-2 and GAPDH blots were from the samegels/blots. These blots were then stripped and reprobed with anti-IRS-2antibody. Data are from a single experiment representative of 4.Locations of 31 kiltodalton (kD), 38 kD, 150 kD, and 225 kD markers areindicated. FIG. 4B is a series of bar graphs showing surface CD107a andintracellular IFN-γ detected by flow cytometry of pIRS-2-specific humanCD8 T-cells following co-culture with indicated human cancer cells.Human cancer cell lines that were HLA-A2-negative are indicated with a*. Data is representative of 3-5 experiments, open boxes: % CD107a⁺;left to right hatched boxes: % IFNγ⁺; right to left hatched boxes: %CD107a⁺/IFNγ⁺. FIG. 4C are immunoblots of pSer¹¹⁰⁰-IRS-2 (top) and totalIRS-2 (bottom) in extracts representing 50 μg total protein of theindicated cancer cells. Location of 160 kD marker is indicated. FIG. 4Dis a graph showing correlation between pSer¹¹⁰⁰-IRS-2 protein and T-cellrecognition of HLA-A2⁺ cancer cells by pIRS-2-specific human CD8 T-cells(data in FIGS. 4A and 4B). Linear regression analyses (solid line) with95% confidence intervals (dashed lines) are shown. Slope issignificantly non-zero.

FIGS. 5A-5C show the results of experiments showingpCDC25b₃₈₋₄₆-specific TCR-expressing human CD8 T-cells recognizedendogenously processed and presented phosphopeptide on human melanomaand breast cancer cells. FIG. 5A is a series of immunoblots showingexpression of total CDC25b (top) and GAPDH (bottom) in indicated cancercells (30 μg total protein from cytoplasmic fraction). 1 μg of HEK293Tcell lysate was loaded in order to not over-expose blot. Representativeblots from two experiments shown. Locations of 30 kD, 40 kD, 60 kD, and80 kD markers are indicated. FIG. 5B is a bar graph showing that surfaceCD107a and intracellular IFN-γ were detected by flow cytometry ofpCDC25b-specific human CD8 T-cells following co-culture with indicatedhuman cancer cells. HLA-A2-negative cancer cells are indicated with a *and dashed line indicates background on HLA-A2^(neg) targets. Datarepresentative of two experiments, open boxes: % CD107a⁺; left to righthatched boxes: % IFNα⁺; right to left hatched boxes: % CD107a⁺/IFNγ⁺.FIG. 5C is a plot showing lack of correlation between CDC25b protein andT-cell recognition of HLA-A2⁺ cancer cells by pCDC25b-specific human CD8T-cells (data in FIGS. 5A and 5B).

FIGS. 6A-6C are a series of immunohistochemistry photos showing thatpSer¹¹⁰⁰-IRS-2 staining was highest in mitotic cancer cells.pSer¹¹⁰⁰-IRS-2 stained tissue sections from the melanoma cell line SLM2(FIG. 6A), ovarian carcinoma OV-90 (FIG. 6B), and a lung melanomametastasis (FIG. 6C) without (left panels) or with (right panels)blocking peptide added to antibody during staining. 100× magnificationis shown. Arrows indicate mitotic cells.

FIGS. 7A-7H are a series of immunohistochemistry photos showingSer¹⁰⁰-phosphorylated IRS-2 expression in metastatic melanoma sectionsinvolving vital organs. pSer¹¹⁰⁰-IRS-2 stained sections (100×magnification) from melanoma metastases in lung, heart and liver (FIGS.7A, 7C, and 7E, respectively), together with the adjacent uninvolvedtissues (FIGS. 7B, 7D, and 7F, respectively). Arrows in FIG. 7A indicatemitotic cells with intense staining; the strongest staining occurs inthe large malignant cells with mitotic figures with the remainder beinga mixture of non-mitotic melanoma cells and peritumoral stroma. Similarincreased staining of mitotically active cells was observed in allmelanoma metastases examined Normal skin specimens stained withpSer¹¹⁰⁰-IRS-2 are shown in FIGS. 7G and 7H). Mild diffuse epidermalstaining for pSer¹¹⁰⁰-IRS-2 is present in FIG. 7G, and only partiallydiminished in the presence of blocking peptide (FIG. 7H).

FIG. 8 is an immunohistochemistry photo showing pSer1100-IRS-2 stainingof colon melanoma metastasis. pSer1100-IRS-2 staining of colorectalcancer containing a melanoma metastasis. Specific staining densities areidentified for the melanoma (3.3×10⁷), peritumoral rectal mucosa(3.1×10⁸), and neighboring colonic mucosa (1.6×10⁷ or 3.4×10⁸).

FIGS. 9A-9C are a series of plots showing enhanced tumor-free survivalfollowing adoptive transfer of phosphopeptide-specific TCR-expressinghuman CD8 T-cells. SLM2AAD melanoma tumor-bearing NOD/SCID/IL-2Rγc^(−/−)mice were injected with phosphopeptide-specific TCR-expressing human CD8T-cells on days 3 and 7, together with 1500 CU/ml IL-2 every other dayfor 10 days. Control animals (open circles) only received IL-2. FIG. 9Ais a plot showing enhanced tumor-free survival following adoptivetransfer of pIRS-2 mTCR phosphopeptide-specific TCR-expressing human CD8T-cells (solid triangles; p=0.0290). FIG. 9B is a plot showing enhancedtumor-free survival following adoptive transfer of pCDC25b mTCRphosphopeptide-specific TCR-expressing human CD8 T-cells (upside downsolid triangles; p=0.0116). FIG. 9C is a plot showing enhancedtumor-free survival following adoptive transfer of combo TCR animals(i.e., animals that received equal amounts of pIRS-2 and pCDC25b-TCRexpressing human CD8 T-cells (solid diamonds; p=0.0116). Tumor freesurvival is equal to the measurement day when the tumor size was >30mm². The p values listed are for Log-rank analysis comparison of controlanimals to experimental group through day 25.

FIGS. 10A-10B depict the results of experiments showing expression ofphosphopeptide-specific murine TCR in human CD8 T-cells conferredrecognition of HLA-A2+ targets and effector function. Human CD8 T-cellswere electroporated with IVT RNA encoding murine TCR αβ chains specificfor pIRS-2 (pIRS2 BK TCR; FIG. 10A; SEQ ID NO: 2) or pDesmuslin (A10(right to left hatched boxes) or A11 (left to right hatched boxes) TCRchains; FIG. 10B), and assayed 12-14 hours later. TCR-expressing humanCD8 T-cells were co-cultured for 18-20 hours with C1R-A2 or C1R-AADpulsed or unpulsed target cells. Supernatants were then harvested andIFN-γ was detected by ELISA. No RNA controls underwent electroporationwith no addition of IVT RNA (open squares).

FIGS. 11A-11C depict the results of experiments showing expression ofpβcatenin-specific murine TCR in human CD8 T-cells conferred recognitionof HLA-A2+ targets and effector function. Human CD8 T-cells wereelectroporated with IVT RNA encoding phosphopeptide-specific murine TCRαβ chains (FIG. 11A: 649 pA10V βcatenin TCR; B: 653 pβcatenin TCR, andassayed 12-14 hours later. FIGS. 11A and 11B: Surface CD107a and/orintracellular IFN-γ were detected by flow cytometry onpβcatenin-specific human CD8 T-cells following co-culture with indicatedC1R-A2 unpulsed or peptide-pulsed targets. x: no stimulators; solidsquares: pβ catenin; open squares: β catenin; solid diamond (FIG. 11A)or solid triangle (FIG. 11B): unpulsed. FIG. 11C shows the comparison ofthe ability of the two pβcatenin-specific TCR chains to recognize theYLDpSGIHSGA (SEQ ID NO: 30) peptide on C1R-A2 pulsed target cells.Notice the 649 pA10V TCR-expressing human CD8 T-cells (solid squares)were able to recognize much lower amounts of pulsed phosphopeptide onthe target cells, suggesting it has a higher affinity than the 653pβcatenin TCR (solid triangles). No RNA controls underwentelectroporation with no addition of IVT RNA (solid circles).

DETAILED DESCRIPTION I. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. Mention of techniques employed hereinare intended to refer to the techniques as commonly understood in theart, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, in some embodiments the phrase “a peptide” refers toone or more peptides.

The term “about”, as used herein to refer to a measurable value such asan amount of weight, time, dose (e.g., therapeutic dose), etc., is meantto encompass in some embodiments variations of ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.1%, in some embodiments ±0.5%, and in some embodiments±0.01% from the specified amount, as such variations are appropriate toperform the disclosed methods.

As used herein, the term “and/or” when used in the context of a list ofentities, refers to the entities being present singly or in any possiblecombination or subcombination. Thus, for example, the phrase “A, B, C,and/or D” includes A, B, C, and D individually, but also includes anyand all combinations and subcombinations of A, B, C, and D.

Throughout the instant disclosure and including in the Figures,phosphorylated amino acids are depicted in lowercase “s”, “t”, or “y”for phosphoserine, phosphothreonine, or phosphotyrosine, respectively.Alternatively, “pS” refers to phosphoserine, “pT” refers tophosphothreonine, and “pY” refers to phosphotyrosine throughout theinstant disclosure and in the Figures.

As used herein, the term “treating” and grammatical variants thereofincluding but not limited to “treatment” and “treat” are used herein torefer to administration of a composition of the presently disclosedsubject matter in order to mitigate a condition in a patient and/or byreducing, inhibiting, and/or eliminating a particular characteristic orevent associated with an undesirable condition including but not limitedto a tumor or a cancer. Thus, the term “treatment” includes preventing acondition from occurring in a patient, particularly when the patient ispredisposed to acquiring the condition; reducing and/or inhibiting thecondition and/or its development and/or progression; and/or amelioratingand/or reversing the condition. Insofar as some embodiments of themethods of the presently disclosed subject matter are directed topreventing conditions, it is understood that the term “prevent” does notrequire that the condition be completely thwarted. Rather, as usedherein, the term “preventing” refers to the ability of one of ordinaryskill in the art to identify a population that is susceptible tocondition, such that administration of the compositions of the presentlydisclosed subject matter might occur prior to onset of the condition.The term does not imply that the condition must be completely avoided.

As used herein, the phrase “effective amount” refers to an amount of acomposition of the presently disclosed subject matter that is sufficientto exhibit a detectable therapeutic effect. The effect is detected by,for example, an improvement in clinical condition, and/or a prevention,reduction, or amelioration of at least one symptom thereof and/or atleast one complication thereof. The precise effective amount for apatient can depend in some embodiments upon the patient's body weight,size, and health; the nature and extent of the condition; and thetherapeutic or combination of therapeutics selected for administration.Therapeutically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of one of ordinary skill in the art (e.g., a clinician).

The term “phosphopeptides” includes MHC class I- and MHC classII-specific phosphopeptides. Exemplary MHC class I phosphopeptides arethe pIRS2₁₀₉₇₋₁₁₀₅ phosphopeptide (SEQ ID NO: 2), the pCDC25b₃₈₋₄₆phosphopeptide (SEQ ID NO: 12), the pDesmuslin₄₂₆₋₄₃₅ phosphopeptide(SEQ ID NO: 19), and the pβ-catenin₃₀₋₃₉ phosphopeptides (SEQ ID NOs: 29and 30). SEQ ID NO: 2 corresponds to amino acids 1097-1105 of a humaninsulin receptor substrate 2 (IRS2) gene product presented as AccessionNo. NP_003740.2 in the GENBANK® biosequence database. SEQ ID NO: 12corresponds to amino acids 38-46 of a human M-phase inducer phosphatase2 isoform 3/CDC25B gene product presented as Accession No. NP_068658.1in the GENBANK® biosequence database. SEQ ID NO: 19 corresponds to aminoacids 426-435 of a human desmuslin/synemin isoform A gene productpresented as Accession No. NP_663780.2 in the GENBANK® biosequencedatabase, and also corresponds to amino acids 426-435 of a humandesmuslin/synemin isoform B gene product presented as Accession No.NP_056101.5 in the GENBANK® biosequence database. SEQ ID NOs: 29 and 30correspond to amino acids 30-39 of a human β-catenin gene productpresented as Accession No. NP_001895.1 in the GENBANK® biosequencedatabase, wherein in SEQ ID NO: 29, the alanine at position 39 ofGENBANK® Accession No. NP_001895.1 is replaced by a valine.

Thus, in some embodiments, the phosphopeptides contain the sequences ofat least one of the MHC class I binding peptides listed in SEQ ID NOs:2, 12, 19, 29, and 30. Moreover, in some embodiments one or more of theserine residues within the recited sequences is phosphorylated. Thephosphorylation can be with a natural phosphorylation (—CH₂—O—PO₃H) orwith an enzyme non-degradable, modified phosphorylation, such as but notlimited to —CH₂—CF₂—PO₃H or —CH₂—CH₂—PO₃H. Some phosphopeptides cancontain more than one of the peptides listed in SEQ ID NOs: 2, 12, 19,29, and 30, for example, if they are overlapping, adjacent, or nearbywithin the native protein from which they are derived.

As used herein, the phrases “proliferative disorder” and “proliferativedisease” refers to a disease, disorder, or condition associated withabnormal and/or undesirable cell proliferation. In some embodiments, aproliferative disease is a cancer, including but not limited to breastcancer, colorectal cancer, squamous carcinoma of the lung, sarcoma,renal cell carcinoma, pancreatic carcinomas, squamous tumors of the headand neck, leukemia, brain cancer, liver cancer, prostate cancer, ovariancancer, and cervical cancer. In some embodiments, the presentlydisclosed compositions and methods are used to treat colorectal cancer,acute myelogenous leukemia (AML), acute lyphocytic leukemia (ALL),chronic lymphocytic lymphoma (CLL), chronic myelogenous leukemia (CML),breast cancer, renal cancer, pancreatic cancer, and/or ovarian cancer.

As used herein, the phrase “specific binding” refers to binding betweena TCR, TCR-like molecule, or portion thereof and an antigen and/or anepitope thereof (including but not limited to a peptide, optionally incomplex with an MHC molecule) that is indicative of the presence of theantigen and/or the epitope thereof. As such, a TCR, TCR-like molecule,or portion thereof is said to “specifically” bind an antigen and/or anepitope thereof when the dissociation constant (Kd) is in someembodiments less than about 1 μM, in some embodiments less that about100 nM, and in some embodiments less than about 10 nM. Interactionsbetween antibodies and antibody-like molecules and an epitope can alsobe characterized by an affinity constant (K_(a)). In some embodiments, aK_(a) of less than about 10⁷/M is considered “high affinity”.

As used herein, the phrase “T cell receptor” and the term “TCR” refer toa surface protein of a T cell that allows the T cell to recognize anantigen and/or an epitope thereof, typically bound to one or more majorhistocompatibilityi complex (MHC) molecules. A TCR functions torecognize an antigenic determinant and to initiate an immune response.Typically, TCRs are heterodimers comprising two different proteinchains. In the vast majority of T cells, the TCR comprises an alpha (α)chain and a beta (β) chain. Approximately 5% of T cells have TCRs madeup of gamma and delta (γ/δ) chains.

TCRs are membrane-anchored heterodimers that are found as part of acomplex with a CD3 chain molecule. Each chain comprises twoextracellular domains: a variable (V) region and a constant (C) region,the latter of which is membrane-proximal. The variable domains ofα-chains and of β-chains consist of three hypervariable regions that arealso referred to as the complementarity determining regions (CDRs). TheCDRs, in particular CDR3, are primarily responsible for contactingantigens and thus define the specificity of the TCR, although CDR1 ofthe α-chain can interact with the N-terminal part of the antigen. CDR1of the β-chain interacts with the C-terminal part of the peptide. TCRsare also characterized by a series of highly conserved disulfide bondsthat link the two chains.

As used herein, the phrase “TCR-like polypeptide” refers to apolypeptide that behaves similarly to a T cell receptor (TCR) in that itspecifically binds to an MHC-bound peptide, optionally an MHC-boundphosphopeptide as disclosed herein. In some embodiments, a “TCR-likeantibody” refers to an antibody, optionally a monoclonal antibody, whichspecifically recognizes an MHC-bound phosphopeptide of the presentlydisclosed subject matter. In some embodiments, such polypeptides aremembers of the Ig Superfamily. In some embodiments, a TCR-likepolypeptide is a single chain TCR (see e.g., U.S. Patent ApplicationPublication No. 2012/0252742; PCT International Patent ApplicationPublication Nos. WO 1996/013593, WO 1999/018129, and WO 2004/056845;U.S. Pat. No. 7,569,664).

As used herein, a “portion” of a TCR or TCR-like polypeptide is asubsequence of a TCR or TCR-like polypeptide that retains a desiredfunction of the TCR or TCR-like polypeptide. In some embodiments, aportion of a TCR or TCR-like polypeptide comprises the domain of the TCRor TCR-like polypeptide that binds to a phosphopeptide/MHC complex(optionally, a phosphopeptide/HLA-A2 complex). Thus, in some embodimentsthe phrase “TCR, TCR-like molecule, or portion thereof” refers to TCRs,TCR-like molecules, and portions thereof that bind to phosphopeptide/MHCcomplexes, including but not limited to phosphopeptide/HLA-A2 complexes.

II. TCRs, TCR-like Molecules, and Portions Thereof, and PhosphopeptideTargets Thereof

In some embodiments, the presently disclosed subject matter providesisolated and/or cloned TCRs, TCR-like molecules, or portions thereofthat bind to post-translationally modified immunogenic therapeutictarget peptides (e.g., phosphopeptides). In some embodiments, a TCR,TCR-like molecule, or portion thereof of the presently disclosed subjectmatter has antigen specificity for an antigen that is characteristic ofa disease or disorder. The disease or disorder can be any disease ordisorder involving an antigen, such as but not limited to an infectiousdisease, an autoimmune disease, or a tumor and/or a cancer.

In some embodiments, the phosphopeptides are fragments oftumor-associated antigens (TAAs; also referred to herein as “cancerantigens”) and/or are TAAs themselves. The phrases “tumor-associatedantigen” and “cancer antigen” as used herein refer to any molecule(e.g., protein, peptide, lipid, carbohydrate, etc.) solely orpredominantly expressed or over-expressed by a tumor cell and/or acancer cell, such that the antigen is associated with the tumor and/orthe cancer. The TAA/cancer antigen additionally can be expressed bynormal, non-tumor, or non-cancerous cells. However, in such a situation,the expression of the TAA/cancer antigen by normal, non-tumor, ornon-cancerous cells is in some embodiments not as robust as theexpression of the TAA/cancer antigen by tumor and/or cancer cells. Thus,in some embodiments the tumor and/or cancer cells overexpress the TAAand/or express the TAA at a significantly higher level as compared tothe expression of the TAA by normal, non-tumor, and/or non-cancerouscells.

The TAA can be an antigen expressed by any cell of any cancer or tumor,including the cancers and tumors described herein. The TAA can be a TAAof only one type of cancer or tumor, such that the TAA is associatedwith or characteristic of only one type of cancer or tumor.Alternatively, the TAA can be characteristic of more than one type ofcancer or tumor. For example, the TAA can be expressed by both breastand prostate cancer cells and not expressed at all by normal, non-tumor,or non-cancer cells.

II.A. Phosphopeptides Based on IRS-2 Gene Products

In some embodiments, a phosphopeptide target is a fragment of an IRS-2gene product. As used herein, “IRS-2” refers to the insulin receptorsubstrate-2 locus and its corresponding gene products. Exemplary IRS-2gene products include the human IRS-2 gene products present in theGENBANK® biosequence database under accession numbers NM_003749.2 (cDNAnucleotide sequence) and NP_003740.2 (amino acid sequence encodedthereby; SEQ ID NO: 1).

IRS proteins are adapter proteins that link signaling from ligand-boundgrowth factor and cytokine receptors, including the insulin receptor,insulin-like growth factor receptor and IL-4 receptor, to multipledownstream SH2-containing signaling proteins to modulate cellulargrowth, metabolism, survival and differentiation (Dearth et al., 2007).IRS-2 is overexpressed at the gene or protein level in pancreatic cancer(Kornmann et al., 1998), hepatocellular carcinoma (Boissan et al.,2005), neuroblastoma (Kim et al., 2004), breast cancer (Jackson et al.,2001), glioblastoma (Knobbe & Reifenberger, 2003), and colorectal cancer(Parsons et al., 2005). IRS-2 overexpression under a mouse mammary tumorvirus promoter causes mammary hyperplasia, tumorigenesis, and metastasis(Jackson et al., 2001; Nagle et al., 2004; Dearth et al., 2006; Chan &Lee, 2008).

The IRS proteins are regulated by phosphorylation of Tyr, Ser, and Thr(Dearth et al., 2007). The breadth of expression of the phosphopeptideamong different cancer cells has not been investigated. It is disclosedherein that phosphorylated IRS-2 is broadly displayed on multiple cancertypes and the resulting phosphopeptide is endogenously processed andpresented at levels that allow strong immune responses to be generatedagainst it. Phosphopeptide-specific CD8⁺ T cells can be generated fromHLA-A2 transgenic mice upon immunization with pIRS-2 phosphopeptides,and these T cells are capable of recognizing and killing human melanomaand breast tumors in vitro and controlling tumor growth in a xenografttumor model system.

In a particular embodiment, a phosphopeptide target that is derived fromthe human IRS-2 protein comprises the amino acid sequence RVASPTSGV (SEQID NO: 2; see also amino acids 1097-1105 of GENBANK® Accession No.NP_003740.2; SEQ ID NO: 1), wherein the serine at position 4 of thissequence is phosphorylated (referred to herein as “the RVApSPTSGVphosphopeptide” and the “IRS-2 phosphopeptide”).

Two TCRs that bind to the IRS-2 phosphopeptide, referred to herein as“IRS-2A” and “IRS-2B”, were isolated as described herein. The nucleotidesequences of the α and β chains and the amino acid sequences encodedthereby for these two IRS-2 phosphopeptide-specific TCRs weredetermined.

For IRS-2A, the α chain nucleotide and amino acid sequences are setforth in SEQ ID NOs: 3 and 4, respectively, and the β chain nucleotideand amino acid sequences are set forth in SEQ ID NOs: 5 and 6,respectively. Analyzing these sequences using the resources availablethrough the website of THE INTERNATIONAL IMMUNOGENETICS INFORMATIONSYSTEM® (www<<dot>>imgt<<dot>>org; hereinafter referred to as “IMGT”)showed that IRS-2A had an alpha chain comprising a V-J region having anamino acid sequence that corresponds to amino acids 20-131 of SEQ ID NO:4, which corresponds to a TRAV9D-4*04 V region and a TRAJ45*01 J region.IRS-2A has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids20-133 of SEQ ID NO: 6, which corresponds to a TRBV14*01 V region, aTRBD1*01 D region, and a TRBJ2-7*01 J region. The beta chain has aTRBC2*03 constant region. Further analysis demonstrated that IRS-2A ischaracterized by an alpha chain comprising a CDR1 region comprisingYSGTPY (amino acids 46-51 of SEQ ID NO: 4), a CDR2 region comprisingYYSGDPVV (amino acids 69-76 of SEQ ID NO: 4), and a CDR3 regioncomprising AVSEGADRLT (amino acids 111-120 of SEQ ID NO: 4); and a betachain comprising a CDR1 region comprising SGHDT (amino acids 46-50 ofSEQ ID NO: 6), a CDR2 region comprising FRDEAV (amino acids 68-73 of SEQID NO: 6), and a CDR3 region comprising ASSLLDSSYEQY (amino acids112-123 of SEQ ID NO: 6).

For IRS-2B, the α chain nucleotide and amino acid sequences are setforth in SEQ ID NOs: 7 and 8, respectively, and the β chain nucleotideand amino acid sequences are set forth in SEQ ID NOs: 9 and 10,respectively. Analyzing these sequences using the IMGT resources showedthat IRS-2B had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 20-132 of SEQ ID NO: 7,which corresponds to a TRAV9D-4*04 V region and a TRAJ44*01 J region.IRS-2B has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids19-131 of SEQ ID NO: 10, which corresponds to a TRBV13-3*01 V region, aTRBD1*01 D region, and a TRBJ2-1*01 J region. The beta chain has aTRBC2*03 constant region. Further analysis demonstrated that IRS-2A ischaracterized by an alpha chain comprising a CDR1 region comprisingYSGTPY (amino acids 46-50 of SEQ ID NO: 8), a CDR2 region comprisingYYSGDPVV (amino acids 69-76 of SEQ ID NO: 8), and a CDR3 regioncomprising AVSAGSGGKLT (amino acids 111-122 of SEQ ID NO: 8); and a betachain comprising a CDR1 region comprising NNHDY (amino acids 45-49 ofSEQ ID NO: 10), a CDR2 region comprising SYVADS (amino acids 67-72 ofSEQ ID NO: 10), and a CDR3 region comprising ASSDRDNYAEQF (amino acids110-122 of SEQ ID NO: 10).

It is understood that the IRS-2A and IRS-2B TCRs disclosed herein areexemplary only, and that other TCRs, TCR-like molecules, and portionsthereof based on the sequence and subsequences of the IRS-2A and IRS-2BTCRs are also encompassed by the presently disclosed subject matter

II.B. Phosphopeptides Based on CDC25b Gene Products

In some embodiments, a phosphopeptide target is a fragment of a CDC25bgene product. As used herein, “CDC25b” refers to the cell division cycle25B locus and its corresponding gene products. The family of CDC25dual-specificity phosphatases regulates the activity of cyclin-dependentkinases by dephosphorylation of Tyr and Thr residues in their activesites (Kiyokawa & Ray, 2008). CDC25b overexpression in multiplemalignancies is correlated with poor prognosis (Kiyokawa & Ray, 2008).However, as with IRS-2, the immunological display of the HLA-A2restricted pCDC25b₃₈₋₄₆ phosphopeptide on different cancer cells has notbeen evaluated. Exemplary CDC25b gene products include the human IRS-2gene products present in the GENBANK® biosequence database underaccession numbers NM_021873.3 (cDNA nucleotide sequence) and NP_068659.1(amino acid sequence encoded thereby; SEQ ID NO: 11). CDC25b is aphosphatase that is required for entry into mitosis. It has beenreported to have oncogenic properties, although the precise role itplays in tumorigenesis and/or carcinogenesis is unknown.

In a particular embodiment, a phosphopeptide target that is derived fromthe human CDC25b protein comprises the amino acid sequence GLLGSPVRA(SEQ ID NO: 11; see also amino acids 38-46 of GENBANK® Accession No.NP_068659.1; SEQ ID NO: 11), wherein the serine at position 5 of thissequence is phosphorylated (referred to herein as “the GLLGpSPVRAphosphopeptide” or the “CDC25b phosphopeptide”).

A TCR that binds to the CDC25b phosphopeptide, referred to herein as“the CDC25b TCR”, was isolated as described herein. The nucleotidesequence of the α and β chains and the amino acid sequences encodedthereby for the CDC25b TCR were determined. The α chain nucleotide andamino acid sequences are set forth in SEQ ID NOs: 13 and 14,respectively, β chain nucleotide and amino acid sequences are set forthin SEQ ID NOs: 15 and 16, respectively.

Analyzing these sequences using the IMGT resources showed that theCDC25b TCR had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 22-132 of SEQ ID NO: 14,which corresponds to a TRAV3-3*02 V region and a TRAJ12*01 J region. TheCDC25b TCR has a TRAC*01 constant region. The beta chain comprises aV-D-J region having an amino acid sequence that corresponds to aminoacids 30-138 of SEQ ID NO: 16, which corresponds to a TRBV13-2*01 Vregion, a TRBD2*01 D region, and a TRBJ2-5*01 J region. The beta chainhas a TRBC2*03 constant region. Further analysis demonstrated that theCDC25b TCR is characterized by an alpha chain comprising a CDR1 regioncomprising DPNSYY (amino acids 48-53 of SEQ ID NO: 14), a CDR2 regioncomprising VFSSTEI (amino acids 71-77 of SEQ ID NO: 14), and a CDR3region comprising AVKPGGYKVV (amino acids 112-121 of SEQ ID NO: 14); anda beta chain comprising a CDR1 region comprising NNHNN (amino acids56-60 of SEQ ID NO: 16), a CDR2 region comprising SYGAGS (amino acids78-83 of SEQ ID NO: 16), and a CDR3 region comprising ASGGDTQY (aminoacids 121-129 of SEQ ID NO: 16). It is understood that the CDC25b TCRdisclosed herein is exemplary only, and that other TCRs, TCR-likemolecules, and portions thereof based on the sequence and subsequencesof the CDC25b TCR are also encompassed by the presently disclosedsubject matter.

II.C. Phosphopeptides Based on Desmuslin Gene Products

In some embodiments, a phosphopeptide target is a fragment of adesmuslin gene product. As used herein, “desmuslin” refers to thedesmuslin locus and its corresponding gene products. Desmuslin is alsoreferred to “synemin”, of which there are multiple isoforms in humans.Exemplary desmuslin gene products include the human gene productspresent in the GENBANK® biosequence database under accession numbersNM_145728.2 (synemin isoform A cDNA nucleotide sequence) and NP_663780.2(amino acid sequence encoded thereby; SEQ ID NO: 17) and NM_015286.5(synemin isoform B cDNA nucleotide sequence) and NP_056101.5 (amino acidsequence encoded thereby; SEQ ID NO: 18).

Desmuslin/synemin proteins are cytoskeletal intermediate filament (IF)proteins that are involved in providing mechanical stress resistance.

In a particular embodiment, a phosphopeptide target that is derived fromthe human desmuslin/synemin protein comprises the amino acid sequenceRTFSPTYGL (SEQ ID NO: 19; see also, for example, amino acids 426-434 ofGENBANK® Accession No. NP_663780.2; SEQ ID NO: 17), wherein the serineat position 4 of this sequence is phosphorylated (referred to herein as“the desmuslin phosphopeptide”).

Two TCRs that bind to the desmuslin phosphopeptide, referred to hereinas “DESA” and “DESB”, were isolated as described herein. The nucleotidesequences of the α and β chains and the amino acid sequences encodedthereby for these two desmuslin phosphopeptide-specific TCRs weredetermined.

For DESA, the α chain nucleotide and amino acid sequences are set forthin SEQ ID NOs: 20 and 21, respectively, and the β chain nucleotide andamino acid sequences are set forth in SEQ ID NOs: 22 and 23,respectively. Analyzing these sequences using the IMGT resources showedthat DESA had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 20-131 of SEQ ID NO: 21,which corresponds to a TRAV9D-4*02 V region and a TRAJ31*01 J region.DESA has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids30-140 of SEQ ID NO: 23, which corresponds to a TRBV12-1*01 V region, aTRBD1*01 D region, and a TRBJ1-1*01/J1-1*02 J region. The beta chain hasa TRBC1*01 constant region. Further analysis demonstrated that DESA ischaracterized by an alpha chain comprising a CDR1 region comprisingYSGTPY (amino acids 46-51 of SEQ ID NO: 21), a CDR2 region comprisingYYSGDPVV (amino acids 69-76 of SEQ ID NO: 21), and a CDR3 regioncomprising VLSYSNNRIF (amino acids 111-120 of SEQ ID NO: 21); and a betachain comprising a CDR1 region comprising SGHSN (amino acids 56-60 ofSEQ ID NO: 23), a CDR2 region comprising HYEKVE (amino acids 78-83 ofSEQ ID NO: 23), and a CDR3 region comprising ASSLGGGEVF (amino acids121-130 of SEQ ID NO: 23).

For DESB, the α chain nucleotide and amino acid sequences are set forthin SEQ ID NOs: 24 and 25, respectively, and the β chain nucleotide andamino acid sequences are set forth in SEQ ID NOs: 26 and 27,respectively. Analyzing these sequences using the IMGT resources showedthat DESB had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 20-132 of SEQ ID NO: 25,which corresponds to a TRAV9D-4*02 V region and a TRAJ56*01 J region.DESB has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids19-128 of SEQ ID NO: 27, which corresponds to a TRBV13-3*01 V region, aTRBD1*01 D region, and a TRBJ2-5*01 J region. The beta chain has aTRBC2*03 constant region. Further analysis demonstrated that DESB ischaracterized by an alpha chain comprising a CDR1 region comprisingYSGTPY (amino acids 46-51 of SEQ ID NO: 25), a CDR2 region comprisingYYSGDPVV (amino acids 69-76 of SEQ ID NO: 25), and a CDR3 regioncomprising VLRYGGNNKLT (amino acids 111-121 of SEQ ID NO: 25), and abeta chain comprising a CDR1 region comprising NNHDY (amino acids 45-49of SEQ ID NO: 27), a CDR2 region comprising SYVADS (amino acids 67-72 ofSEQ ID NO: 27), and a CDR3 region comprising ASRYRDTQY (amino acids110-118 of SEQ ID NO: 27).

It is understood that the DESA and DESB TCRs disclosed herein areexemplary only, and that other TCRs, TCR-like molecules, and portionsthereof based on the sequence and subsequences of the DESA and DESB TCRsare also encompassed by the presently disclosed subject matter.

II.D. Phosphopeptides Based on β-catenin Gene Products

In some embodiments, a phosphopeptide target is a fragment of aβ-catenin gene product. As used herein, “β-catenin” refers to the CTNNB1locus and its corresponding gene products. Exemplary β-catenin geneproducts include the human gene products present in the GENBANK®biosequence database under accession numbers NM_001904.3 (cDNAnucleotide sequence) and NP_001895.1 (amino acid sequence encodedthereby; SEQ ID NO: 28).

β-catenin proteins are dual function proteins that are involved inregulating the coordination of cell-cell adhesion and genetranscription. Mutations and overexpression of β-catenin have beenassociated with hepatocellular carcinoma, colorectal carcinoma, lungcancer, malignant breast tumors, ovarian cancer, and endometrial cancer(Morin, 1999). β-catenin is regulated by the β-catenin destructioncomplex, and in particular by the adenomatous polyposis coli (APC)protein, encoded by the tumor-suppressing APC gene. Genetic mutation ofthe APC gene is also strongly linked to cancers, and in particularcolorectal cancer resulting from familial adenomatous polyposis (FAP).

In particular embodiments, phosphopeptide targets that are derived fromthe human β-catenin protein comprise the amino acid sequences YLDSGIHSGV(SEQ ID NO: 29; see also, for example, amino acids 30-39 of GENBANK®Accession No. NP_001895.1 (SEQ ID NO: 28), wherein the alanine atposition 39 of GENBANK® Accession No. NP_001895.1 is replaced by avaline), wherein the serine at position 4 of this sequence isphosphorylated (referred to herein as “the β-catenin phosphopeptide A”);and YLDSGIHSGA (SEQ ID NO: 30; see also, for example, amino acids 30-39of GENBANK® Accession No. NP_001895.1; SEQ ID NO: 28), wherein theserine at position 4 of this sequence is phosphorylated (referred toherein as “the β-catenin phosphopeptide B”).

TCRs that bind to the β-catenin phosphopeptides A and B, referred toherein as “βCATA” and “βCATB”, respectively, were isolated as describedherein. The nucleotide sequences of the α and β chains and the aminoacid sequences encoded thereby for these two β-cateninphosphopeptide-specific TCRs were determined.

For βCATA, the α chain nucleotide and amino acid sequences are set forthin SEQ ID NOs: 31 and 32, respectively, and the β chain nucleotide andamino acid sequences are set forth in SEQ ID NOs: 33 and 34,respectively. Analyzing these sequences using the IMGT resources showedthat βCATA had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 21-129 of SEQ ID NO: 32,which corresponds to a TRAV13*02 V region and a TRAJ58*01 J region.βCATA has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids20-128 of SEQ ID NO: 34, which corresponds to a TRBV5*01 V region, aTRBD1*01 D region, and a TRBJ2-7*01 J region. The beta chain has aTRBC2*03 constant region. Further analysis demonstrated that βCATA ischaracterized by an alpha chain comprising a CDR1 region comprisingSTATR (amino acids 47-51 of SEQ ID NO: 32), a CDR2 region comprisingNPSGT (amino acids 69-73 of SEQ ID NO: 32), and a CDR3 region comprisingAIPPGTGSKLS (amino acids 108-118 of SEQ ID NO: 32), and a beta chaincomprising a CDR1 region comprising LGHNA (amino acids 45-49 of SEQ IDNO: 34), a CDR2 region comprising YNLKQL (amino acids 67-72 of SEQ IDNO: 34), and a CDR3 region comprising ASSQGQKGY (amino acids 110-118 ofSEQ ID NO: 34).

For βCATB, the α chain nucleotide and amino acid sequences are set forthin SEQ ID NOs: 35 and 36, respectively, and the β chain nucleotide andamino acid sequences are set forth in SEQ ID NOs: 37 and 38,respectively. Analyzing these sequences using the IMGT resources showedthat βCATB had an alpha chain comprising a V-J region having an aminoacid sequence that corresponds to amino acids 21-130 of SEQ ID NO: 36,which corresponds to a TRAV8D-2*02 V region and a TRAJ34*02 J region.βCATB has a TRAC*01 constant region. The beta chain comprises a V-D-Jregion having an amino acid sequence that corresponds to amino acids20-129 of SEQ ID NO: 38, which corresponds to a TRBV5*01 V region, aTRBD2*01 D region, and a TRBJ2-1*01 J region. The beta chain has aTRBC2*03 constant region. Further analysis demonstrated that βCATB ischaracterized by an alpha chain comprising a CDR1 region comprisingTYTTV (amino acids 47-51 of SEQ ID NO: 36), a CDR2 region comprisingIRSNERE (amino acids 69-75 of SEQ ID NO: 36), and a CDR3 regioncomprising ATGPNTNKVV (amino acids 110-119 of SEQ ID NO: 36), and a betachain comprising a CDR1 region comprising LGHKA (amino acids 45-49 ofSEQ ID NO: 38), a CDR2 region comprising YNLKQL (amino acids 67-72 ofSEQ ID NO: 38), and a CDR3 region comprising ASSQGGAEQF (amino acids110-119 of SEQ ID NO: 38).

It is understood that the βCATA and βCATB TCRs disclosed herein areexemplary only, and that other TCRs, TCR-like molecules, and portionsthereof based on the sequence and subsequences of the βCATA and βCATBTCRs are also encompassed by the presently disclosed subject matter.

II.E. Modifications to TCR Sequences to Generate Soluble TCRs, TCR-LikeMolecules, and Portions Thereof

In some embodiments, the TCRs, TCR-like molecules, and portions thereofof the presently disclosed subject matter are modified to generatesoluble TCRs, TCR-like molecules, and portions thereof. Exemplarymethods for generating soluble TCRs are disclosed, for example, in PCTInternational Patent Application Publication No. WO 1998/39482; U.S.Patent Application Publication No. 2005/0214284, and U.S. PatentApplication Publication No. 2011/0070191.

By way of example and not limitation, a TCR, TCR-like molecule, orportion thereof of the presently disclosed subject matter can bemodified to generate a soluble derivative thereof by deleting orotherwise mutating some or all of the amino acids of the transmembraneregion and/or the cytoplasmic tail. In some embodiments, at least one ofand in some embodiments in both of the α and β chain sequences aremutated and/or deleted. For the TCRs, TCR-like molecules, and portionsthereof of the presently disclosed subject matter referred to hereinabove in Sections II.A.-II.D., a soluble TCR, TCR-like molecule, or aportion thereof comprises in some embodiments CDR1, CDR2, CDR3, or anycombination thereof of any of the polypeptides of SEQ ID NOs: 4, 6, 8,10, 14, 16, 21, 23, 25, 27, 32, 34, 36, or 38. In some embodiments, CDRsare as set forth in Table 1 below.

TABLE 1 CDR Sequences SEQ ID CDR1 CDR2 CDR3 NO. (amino acids)(amino acids) (amino acids)  4 YSGTPY; YYSGDPVV; AVSEGADRLT; (46-51)(69-76) (111-120)  6 SGHDT FRDEAV ASSLLDSSYEQY (46-50) (68-73) (112-123) 8 YSGTPY YYSGDPVV AVSAGSGGKLT (46-50) (69-76) (111-122) 10 NNHDY SYVADSASSDRDNYAEQF (45-49) (67-72) (110-122) 14 DPNSYY VFSSTEI AVKPGGYKVV(48-53) (71-77) (112-121) 16 NNHNN SYGAGS ASGGDTQY (56-60) (78-83)(121-129) 21 YSGTPY YYSGDPVV VLSYSNNRIF (46-51) (69-76) (111-120) 23SGHSN HYEKVE ASSLGGGEVF (56-60) (78-83) (121-130) 25 YSGTPY YYSGDPVVVLRYGGNNKLT (46-51) (69-76) (111-121) 27 NNHDY SYVADS ASRYRDTQY (45-49)(67-72) (110-118) 32 STATR NPSGT AIPPGTGSKLS (47-51) (69-73) (108-118)34 LGHNA YNLKQL ASSQGQKGY (45-49) (67-72) (110-118) 36 TYTTV IRSNEREATGPNTNKVV (47-51) (69-75) (110-119) 38 LGHKA YNLKQL ASS QGGAEQF (45-49)(67-72) (110-119)

In some embodiments, single-chain (“sc”) constructs such as thosedisclosed in U.S. patent application Ser. Nos. 08/813,781 and 08/943,086can be employed. Briefly, a single-chain (“sc”) TCR molecule includesV-α and V-β chains that are covalently linked through a suitable linkersequence. For example, the V-α chain can be covalently linked to the V-βchain through a linker sequence (optionally a peptide linker sequence)fused to the C-terminus of the V-α chain and the N-terminus of the V-βchain. The V-α and V-β chains of the sc-TCR fusion protein are in someembodiments about 200 to 400 amino acids in length, in some embodimentsabout 300 to 350 amino acids in length, and in some embodiments can beat least 90%, 95%, 97%, 98%, 99%, or 100% identical to the V-α and V-βchains of the presently disclosed TCRs, TCR-like molecules, and portionsthereof.

As disclosed in U.S. patent application Ser. No. 08/943,086 application,the V-α chain of a sc-TCR molecule can in some embodiments furtherinclude a C-β chain or fragment thereof fused to the C-terminus of theV-β chain. Further, the V-α chain can in some embodiments include a C-αchain or fragment thereof fused to the C-terminus of the V-α chain andthe N-terminus of the linker sequence.

As further disclosed in U.S. patent application Ser. No. 08/943,086,additional sc-TCR proteins of the presently disclosed subject matterinclude for example two peptide linker sequences, where the firstpeptide linker sequence is fused between the C-terminus of the V-α chainand the N-terminus of the V-β chain. The C-terminus of the V-β chain canbe fused to the N-terminus of a C-β chain fragment. The second peptidelinker is then fused to the C-terminus of the V-β chain or C-β chainfragment. In some embodiments, sc-TCR proteins can be made by fusing theV-β chain to the V-α chain through a suitable peptide linker in whichthe C-terminus of the V-β chain or C-β chain fragment thereof and theN-terminus of the V-α chain are covalently linked.

Thus, in some embodiments the TCRs, TCR-like molecules, and portionsthereof of the presently disclosed subject matter are soluble TCRcytoplasmic domains, TCR-like cycloplasmic domains, and portions thereofthat are stable at low concentrations and which can recognizeMHC-peptide complexes. See e.g., U.S. Patent Application Publication No.2002/0119149, which is incorporated by reference.

II.F. Conjugates of TCRs, TCR-Like Molecules, and Portions Thereof

In some embodiments, the TCRs, TCR-like molecules, and portions thereofof the presently disclosed subject matter (optionally wherein the TCRs,TCR-like molecules, and portions thereof are soluble TCRs, TCR-likemolecules, and portions thereof) can be conjugated to one or more activeagents. As used herein, the phrase “active agent” refers to any moleculethat imparts to a TCR, TCR-like molecule, or portion thereof an activityof interest (including but not limited to a biological activity) thatthe TCR, TCR-like molecule, or portion thereof would not have absent theactive agent. Exemplary active agents include immunostimulatory peptidesand/or proteins, detectable labels, other TCRs, other TCR-likemolecules, and/or portions thereof etc.

II.F.1. Immunostimulatory Peptides and Proteins

In some embodiments, the active agents comprise immunostimulatorypeptides and/or proteins, and/or moieties such as but not limited to CD3agonists (e.g., anti-CD3 antibodies). The CD3 antigen is present onmature human T cells, thymocytes, and a subset of natural killer cells.It is associated with the TCR and is involved in signal transduction ofthe TCR. Antibodies specific for the human CD3 antigen are well known.One such antibody is the murine monoclonal antibody OKT3 which was thefirst monoclonal antibody approved by the FDA. OKT3 is reported to be apotent T cell mitogen (Van Wauwe, 1980; U.S. Pat. No. 4,361,539) and apotent T cell killer (Wong et al., 1990). Other antibodies specific forthe CD3 antigen have also been reported (see PCT International PatentApplication Publication No. WO 2004/106380; U.S. Patent ApplicationPublication No. 2004/0202657; U.S. Pat. Nos. 6,750,325; 6,706,265; GreatBritain Patent Publication GB 2249310A; Clark et al., 1989; U.S. Pat.No. 5,968,509; U.S. Patent Application Publication No. 2009/0117102)Immune mobilising mTCR Against Cancer (ImmTAC; Immunocore Limited,Milton Park, Abington, Oxon, United Kingdom) are bifunctional proteinsthat combine affinity monoclonal T cell receptor (mTCR) targeting with atherapeutic mechanism of action (i.e., an anti-CD3 scFv).

II.F.2. Detectable Labels

Other suitable tags for detectably-labeling the TCRs, TCR-likemolecules, and/or portions thereof include biotin, streptavidin, a celltoxin of, e.g., plant or bacterial origin such as, e.g., diphtheriatoxin (DT), shiga toxin, abrin, cholera toxin, ricin, saporin,pseudomonas exotoxin (PE), pokeweed antiviral protein, or gelonin.Biologically active fragments of such toxins are well known in the artand include, e.g., DT A chain and ricin A chain. Additionally, the toxincan be an agent active at the cell surface such as, e.g., phospholipaseenzymes (e.g., phospholipase C). See e.g., Moskaug et al., 1989; Pastanet al., 1986; Pastan et al., 1992; Olsnes & Pihl, 1981; PCTInternational Patent Application Publication No. WO 1994/29350; PCTInternational Patent Application Publication No. WO 1994/04689; and U.S.Pat. No. 5,620,939 for disclosure relating to making and using proteinscomprising effectors or tags. An example of a tag that performs a biotinacceptor function is a BirA tag, as described in Beckett et al., 1999.As further described in Examples below, a BirA tag sequence can beincluded in a TCR, TCR-like molecule, and/or a portion thereof topromote biotinylation of the protein. Further, a tag can be achemotherapeutic drug such as, e.g., vindesine, vincristine, vinblastin,methotrexate, adriamycin, bleomycin, or cisplatin.

Additionally, a tag can be a radionuclide or chelate, suitable fordiagnostic or imaging studies such as iodine-131, yttrium-90,rhenium-188, iodine-123, indium-111, technetium-99m, gallium-67,thallium-201, or bismuth-212. Among the radionuclides used,gamma-emitters, positron-emitters, x-ray emitters andfluorescence-emitters are suitable for localization, while beta-emittersand alpha-emitters may also be used. Other suitable radioisotopes forthe methods of the present invention include but are not limited to,cadmium-109, actinium-225, actinium-227, astatine-211, iodine-125,iodine-126, iodine-133, dysprosium-165, dysprosium-166, bismuth-212,bismuth-213, bromine-77, indium-113m, gallium-67, gallium-68,ruthenium-95, ruthenium-97, ruthenium-101, ruthenium-103, ruthenium-105,mercury-107, mercury-203, rhenium-186, rhenium-188, tellurium-99m,tellurium-121m, tellurium-122m, tellurium-125m, thulium-165,thulium-167, thulium-168, fluorine-18, silver-11, platinum-197,palladium-109, copper-67, phosphorus-32, phosphorus-33, yttrium-90,scandium-47, samarium-153, lutetium-177, rhodium-105, praseodymium-142,praseodymium-143, promethium-149, terbium-161, holmium-166, gold-198,gold-199, cobalt-57, cobalt-58, chromium-51, iron-59, selenium-75, andytterbium-169. Preferably the radioisotope will emit in the 10-5,000 keyrange, more preferably 50-1,500 key, most preferably 50-500 key.

Suitable positron emitters and other useful radionuclides include, butare not limited to, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁵¹Mn, ⁵²Fe, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu,⁶²Cu, ⁶⁴Cu, ⁶²Zn, ⁶³Zn, ⁷⁰As, ⁷¹As, ⁷²As, ⁷⁶Br, ⁸²Rb, ⁸⁶Y, ⁸⁹Zr,^(94m)Tc, ¹¹⁰In, ¹²⁰I, ¹²⁴I, ¹²²Xe, ¹²⁸Ba, ¹³¹Ba, ⁷Be, ²⁰⁴Bi, ²⁰⁵Bi,²⁰⁶Bi, ¹⁴C, ³⁶Cl, ⁴⁸Cr, ⁵¹Cr, ¹⁵⁵Eu, ¹⁵³Gd, ⁶⁶Ga, ⁷²Ga, ³H, ^(115m)In,¹⁸⁹Ir, ^(191m)Ir, ¹⁹²Ir, ¹⁹⁴Ir, ⁵⁵Fe, ^(119m)Os, ⁴²K, ²²⁶Ra, ¹⁸⁶Re,¹⁸⁸Re, ^(82m)Rb, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ₁₀₅Ag, ²²Na, ²⁴Na, ⁸⁹Sr, ³⁵S, ³⁸S,¹⁷⁷Ta, ⁹⁶Tc, ²⁰¹Tl, ²⁰²Tl, ¹¹³Sn, ^(117m)Sn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁷⁴Yb, ⁸⁸Y,⁹⁰Y, ⁶²Zn, and ⁶⁵Zn.

Suitable chelates include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7-tricarboxymethyl-1,4,7,10 teraazacyclododecanetriacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-C1-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5 sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylenediaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (O and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM).

Other suitable tags include polyhistidine, fluorescent label,chemiluminescent label, nuclear magnetic resonance active label,chromophore label, positron emitting isotope detectable by a positronemission tomography (“PET”) scanner, enzymatic markers such asbeta-galactosidase and peroxidase including horse radish peroxidase, ananoparticle, a paramagnetic metal ion, a contrast agent or an antigenictag.

A suitable fluorescent label could include, but is not limited to, a¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodaminelabel, a phycoerythrin label, a phycocyanin label, an allophycocyaninlabel, an o-phthaldehyde label, a Texas Red label, a fluorescaminelabel, a lanthanide phosphor label, a fluorescent protein label, forexample a green fluorescent protein (GFP) label, or a quantum dot label.Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, an aequorin label, etc.

Suitable paramagnetic metal ions include, but are not limited to, Mn²⁺,Cu²⁺, Fe²⁺, Co²⁺, Ni²⁺, Gd³⁺, Eu³⁺, Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺, Fe³⁺, Ti³⁺,Tb³⁺, Nd³⁺, Sm³⁺, Ho³⁺, Er³⁺, Pa⁴⁺, and Eu²⁺.

Enzyme markers that may be used include any readily detectable enzymeactivity or enzyme substrate. Such enzymes include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, alcoholdehydrogenase, glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,luciferase, and DNA polymerase.

II.F.3. Conjugates with Other TCRs, TCR-Like Molecules, and PortionsThereof: Multivalent and Multimeric TCRs, TCR-Like Molecules, andPortions Thereof

The soluble TCRs, TCR-like molecules, and portions thereof of thepresently disclosed subject matter include monomeric and multimericTCRs, TCR-like molecules, and portions thereof. Multimeric TCRs,TCR-like molecules, and portions thereof include those in which the TCR,TCR-like molecule, or portion thereof is fused to polypeptide domains ortags that facilitate multimerization. Such domains includeimmunoglobulin, leucine zipper, helix-turn-helix, and barrel-barrelmotifs that facilitate protein dimerization. Such tags includeantibody-binding epitopes, streptavidin-binding peptides, 6×His motif,biotin ligase target motif, and the like. Multimeric TCRs, TCR-likemolecules, or portions thereof also include those generated throughchemically crosslinking reactive amino acids or polysaccharides. Suchamino acids (or polysaccharides) can be inherent in the structure of theTCRs, TCR-like molecules, and portions thereof, or can be added throughgenetic modification. Multimeric TCRs, TCR-like molecules, and portionsthereof also include those generated through attachment to anothermolecule (or molecules) that may or may not include a detectable labelas described herein. Such attachment molecules include streptavidin,biotin, antibodies, protein A or scaffolds that include protein-, lipid-and polysaccharide-coated or uncoated beads, nanoparticles, solid-phasesurfaces, arrays, matrices, as described. For example, in variousembodiments in which the detectable label is biotin, the method furthercomprises combining the TCR, TCR-like molecule, and/or portion thereofwith streptavidin to multimerize the TCR, TCR-like molecule, and/orportion thereof.

It will be appreciated that any one of the tags disclosed herein can beused to detectably label the TCRs of the presently disclosed subjectmatter, particularly to detect cells and/or tissues such as, but notlimited to tumor and/or cancer cells and tissues, expressing aphosphopeptide target of interest.

II.G. Substantially Identical TCRs, TCR-Like Molecules, and PortionsThereof

In some embodiments, a TCR, TCR-like molecule, or portion thereof of thepresently disclosed subject matter comprises a nucleotide or amino acidsequence that is substantially identical to any of SEQ ID NOs: 3-10,13-16, 20-27, and 31-38.

The term “substantially identical”, as used herein to describe a degreeof similarity between nucleotide or amino acid sequences, refers to twoor more sequences that have in some embodiments at least about least60%, in some embodiments at least about 70%, in some embodiments atleast about 80%, in some embodiments at least about 90%, in someembodiments at least about 95%, in some embodiments at least about 96%,in some embodiments at least about 97%, in some embodiments at leastabout 98%, and in some embodiments at least about 99% nucleotide oramino acid identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm as setforth herein below or by visual inspection. The substantial identityexists in nucleotide or amino acid sequences of in some embodiments atleast about 25 residues, in some embodiments at least about 50 residues,in some embodiments at least about 100 residues, in some embodiments atleast about 150 residues, in some embodiments at least about 200residues, in some embodiments at least about 500 residues, in someembodiments at least about 1000 residues, and in some embodiments innucleotide or amino acid sequences comprising a full length of any ofSEQ ID NOs: 3-10, 13-16, 20-27, and 31-38. The term “full length”, asused herein refers to the complete nucleotide or amino acid sequence ofany of these particular SEQ ID NOs.

Thus, the terms “identical” or percent “identity” in the context of twoor more nucleotide or amino acid sequences refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms disclosed herein or by visualinspection.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer program, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are selected. The sequencecomparison algorithm then calculates the percent sequence identity forthe designated test sequence(s) relative to the reference sequence,based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, 1981, by the homologyalignment algorithm of Needleman & Wunsch, 1970, by the search forsimilarity method of Pearson & Lipman, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group,Madison, Wis.), or by visual inspection. See generally, Ausubel et al.,1995.

A preferred algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described byAltschul et al., 1990. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (www<<dot>>ncbi<<dot>>nlm<<dot>>nih<<dot>>gov). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff & Henikoff, 1992.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See e.g., Karlin & Altschul, 1993. One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

III. Nucleic Acids

III.A. Nucleic Acids Encoding the Presently Disclosed TCRs, TCR-LikeMolecules, and Portions Thereof

In some embodiments, the presently disclosed subject matter providesnucleic acids that encode the presently disclosed TCRs, TCR-likemolecules, and portions thereof. Exemplary nucleic acids that encode thedisclosed TCRs, TCR-like molecules, and portions thereof include SEQ IDNOs: 3-10, 13-16, 20-27, and 31-38 as well as subsequences thereof.

As used herein, the phrases “nucleic acid”, “polynucleotide”,“oligonucleotide”, and “nucleic acid molecule” are used interchangeablyto refer to a polymer of DNA and/or RNA, which can be single-stranded,double-stranded, or multi-stranded, synthesized or obtained (e.g.,isolated and/or purified) from natural sources, which can containnatural, non-natural, and/or altered nucleotides, and which can containnatural, non-natural, and/or altered internucleotide linkages including,but not limited to phosphoroamidate linkages and/or phosphorothioatelinkages instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

In some embodiments, the nucleic acids of the presently disclosedsubject matter comprise a nucleotide sequence as set forth in any of SEQID NOs: 3-10, 13-16, 20-27, and 31-38 as well as subsequences thereof.In some embodiments, the nucleotide sequence does not comprise anyinsertions, deletions, inversions, and/or substitutions relative to SEQID NOs: 3-10, 13-16, 20-27, and 31-38, although contiguous subsequencesof any of these SEQ ID NOs. is encompassed within the presentlydisclosed subject matter. In some embodiments, however, a nucleotidesequence can comprise one or more insertions, deletions, inversions,and/or substitutions relative to SEQ ID NOs: 3-10, 13-16, 20-27, and31-38.

In some embodiments, the nucleic acids of the presently disclosedsubject matter are recombinant. As used herein, the term “recombinant”refers to (i) molecules that are constructed outside living cells byjoining natural or synthetic nucleic acid segments to nucleic acidmolecules that can replicate in a living cell; or (ii) molecules thatresult from the replication of those described in (i) above. Forpurposes herein, the replication can be in vitro replication or in vivoreplication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art (seee.g., Sambrook & and Russell, 2001; and Ausubel et al., 1989). Forexample, a nucleic acid can be chemically synthesized using naturallyoccurring nucleotides and/or variously modified nucleotides designed toincrease the biological stability of the molecules and/or to increasethe physical stability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester,3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.Alternatively or in addition, one or more of the nucleic acids of thepresently disclosed subject matter can be purchased from a commercialsource such as, but not limited to Macromolecular Resources of FortCollins, Colo. and Synthegen of Houston, Tex.

The nucleic acid can comprise any nucleotide sequence that encodes anyof the modified TCRs, TCR-like molecules, portions thereof,polypeptides, proteins, or functional portions or functional variantsthereof of the presently disclosed subject matter. For example, in someembodiments the nucleic acid can comprise a nucleotide sequencecomprising SEQ ID NOs: 3, 5, 7, 9, 13, 15, 20, 22, 24, 26, 31, 33, 35,or 37, and/or can encode a polypeptide having an amino acid sequence asset forth in SEQ ID NOs: 4, 6, 8, 10, 14, 16, 21, 23, 25, 27, 32, 34,36, or 38, or a portion thereof. The nucleotide sequence can in someembodiments comprise a nucleotide sequence which is degenerate to any ofthese sequences or a combination of degenerate sequences.

The presently disclosed subject matter also provides an isolated orpurified nucleic acid comprising a nucleotide sequence that iscomplementary to the nucleotide sequence of any of the nucleic acidsdescribed herein or a nucleotide sequence that hybridizes understringent conditions to the nucleotide sequence of any of the nucleicacids described herein. In some embodiments, the nucleotide sequencethat hybridizes under stringent conditions hybridizes under highstringency conditions. As used herein, the phrase “high stringencyconditions” refers to a set of hybridization condition wherein anucleotide sequence specifically hybridizes to a target sequence (e.g.,a nucleotide sequence of any of the nucleic acids described herein) inan amount that is detectably stronger than non-specific hybridization.High stringency conditions include conditions that would distinguish apolynucleotide with an exact complementary sequence, or one containingonly a few scattered mismatches, from a random sequence that happened tohave a few small regions (e.g., 3-10 bases) that matched the nucleotidesequence. Such small regions of complementarity are more easily meltedthan a full-length complement of 14-17 or more bases, and highstringency hybridization makes them easily distinguishable. Relativelyhigh stringency conditions would include, for example, low salt and/orhigh temperature conditions, such as provided by about 0.02-0.1 M NaClor the equivalent, at temperatures of about 50-70° C. Such highstringency conditions tolerate little, if any, mismatch between anucleotide sequence and a target, and are particularly suitable fordetecting expression of any of the TCRs, TCR-like molecules, or portionsthereof described herein. It is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide. An exemplary high stringency hybridization condition employs0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate (SDS) at 50° C. (0.1×SSC/0.1% SDS). Denaturing agents, such asformamide, can also be employed. Exemplary high stringency hybridizationconditions employing formamide include 50% (v/v) formamide with 0.1%bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50-100 μg/ml), 0.1%SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C. followed bya high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

III.B. Vectors for Recombinant Expression of the Presently DisclosedTCRs, TCR-Like Molecules, and Portions Thereof

The nucleic acids of the presently disclosed subject matter can in someembodiments be incorporated into a vector, optionally an expressionvector, further optionally a recombinant expression vector. Thepresently disclosed subject matter thus provides in some embodimentsrecombinant expression vectors comprising any of the nucleic acidsdisclosed herein. As sued herein, the phrases “expression vector” and“recombinant expression vector” refer to genetically-modifiedoligonucleotide and/or polynucleotide constructs that permit theexpression of an mRNA, protein, polypeptide, and/or peptide by a hostcell, when the construct comprises a nucleotide sequence encoding themRNA, protein, polypeptide, and/or peptide, and the vector is contactedwith the cell under conditions sufficient to have the mRNA, protein,polypeptide, and/or peptide expressed within the cell. The vectors ofthe presently disclosed subject matter are in some embodiments notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. Expression vectors can comprise any type ofnucleotides, including, but not limited to DNA and RNA, which can besingle-stranded or double-stranded, synthesized or obtained in part fromnatural sources, and which can contain natural, non-natural, and/oraltered nucleotides. The expression vectors can comprisenaturally-occurring and/or non-naturally-occurring internucleotidelinkages. In some embodiments, non-naturally occurring or alterednucleotides or internucleotide linkages do not hinder the transcriptionor replication of the vector.

The expression vectors of the presently disclosed subject matter can beany suitable expression vector, and can be used to transform ortransfect any suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. In some embodiments, the vector can be selected from thegroup consisting of the pUC series (Fermentas Life Sciences), thepBluescript series (Stratagene, LaJolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophagevectors, such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149,also can be used. Examples of plant expression vectors include pBI01,pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech).

In some embodiments, the recombinant expression vector is a viralvector, including but not limited both integrating and non-integratingviral vectors. Exemplary viral vectors include, but are not limited toadenoviral vectors, lentiviral vectors, retroviral vectors, episomalvectors, and non-episomal vectors. Exemplary viral vectors are disclosedin, for example, U.S. Pat. Nos. 8,119,772 and 8,552,150, both to Yang etal.; U.S. Pat. Nos. 6,277,633 and 6,521,457, both to Olsen; and U.S.Patent Application Publication No. 2012/0135034 of Dropulic and U.S.Patent Application Publication No. 2008/0254008 of Dropulic et al. (bothassigned to Lentigen Corporation of Gaithersburg, Md., United States ofAmerica). Lentiviral vector systems are also commercially available fromCell Biolabs, Inc. of San Diego, Calif., United States of America andOriGene Technologies, Inc. of Rockville, Md., United States of America.In some embodiments, a vector is a viral episomal vector, optionallybased on adenovirus and/or adeno-associated virus (AAV). An exemplaryminimal adenovirus-based episomal vector is described in PCTInternational Patent Application Publication No. WO 2002/085287 ofBalague et al. A non-viral episomal vector is disclosed in WO1998/007876 of Antoniou et al.

The expression vectors of the presently disclosed subject matter can beprepared using standard recombinant DNA techniques described in, forexample, Sambrook & Russell, 2001; Ausubel et al., 1989. Constructs ofexpression vectors, which can be circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

In some embodiments, an expression vector comprises regulatorysequences, including but not limited to transcription, translation,initiation, and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant, or animal) into which the vectoris to be introduced, as appropriate and taking into considerationwhether the vector is DNA- or RNA-based.

An expression vector of the presently disclosed subject matter can alsoinclude one or more marker genes, which allow for selection oftransformed or transfected hosts. Marker genes can include biocideresistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike. Suitable marker genes for an expression vectors can include, forexample, neomycin/G418 resistance genes, hygromycin resistance genes,histidinol resistance genes, tetracycline resistance genes, andampicillin resistance genes.

An expression vector can comprise a native or non-native promoteroperably linked to the nucleotide sequence encoding the modified TCR,TCR-like molecule, portion thereof, polypeptide, or protein (includingfunctional portions and functional variants thereof), or to thenucleotide sequence that is complementary to or that hybridizes to anucleotide sequence encoding the modified TCR, TCR-like molecule,portion thereof, polypeptide, or protein disclosed herein. The selectionof promoters, in some embodiments strong, weak, inducible,tissue-specific, and/or developmental-specific, is within the ordinaryskill of the artisan. Similarly, the combining of a nucleotide sequencewith a promoter is also within the skill of the artisan. The promotercan be in some embodiments a non-viral promoter or a viral promoterincluding, but not limited to a cytomegalovirus (CMV) promoter, an SV40promoter, an RSV promoter, a promoter found in the long-terminal repeatof a retrovirus, etc.

An expression vector can in some embodiments be designed for transientexpression, stable expression, or both transient and stable expression.Also, an expression vector can be made for constitutive expression orfor inducible expression.

Further, expression vectors can in some embodiments be made to include asuicide gene. As used herein, the phrase “suicide gene” refers to anucleotide sequence that causes a cell expressing the nucleotidesequence to die. A suicide gene can in some embodiments be a nucleotidesequence that confers sensitivity upon a cell expressing the nucleotidesequence as a transcription product and/or as a translation product toan agent (such as but not limited to a drug) such that when the cell iscontacted with and/or exposed to the agent, the agent directly orindirectly causes the cell to die. Suicide genes are known in the artand include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase (see e.g., Springer, 2004).

IV. Host Cells for Production and/or Expression of TCRs, TCR-LikeMolecules, and Portions Thereof

In some embodiments, the presently disclosed subject matter alsoprovides host comprising the disclosed isolated and/or soluble TCR,TCR-like molecule, or portion thereof, and/or an isolated nucleic aciddisclosed herein. In some embodiments, the host cells are employed forthe product and/or expression of a disclosed isolated and/or solubleTCR, TCR-like molecule, or portion thereof.

V. Isolation of Soluble TCRs, TCR-Like Molecules, and Portions Thereof

In some embodiments, the TCRs, TCR-like molecules, and the portionsthereof are soluble. As used herein, the term “soluble” refers to thefact that a TCR, TCR-like molecule, or a portion thereof is not anchoredto a cell membrane via a transmembrane region as is typical for fulllength TCRs. Methods for producing and isolating soluble TCRs, TCR-likemolecules, and portions thereof are exemplified in Molloy et al., 2005;Fremont et al., 1996; Pecorari et al., 1999; and U.S. Patent ApplicationPublication No. 2005/0214284 of Price-Schiavi et al.

In some embodiments, soluble TCRs, TCR-like molecules, and portionsthereof are produced by screening a phage library. An exemplary methodfor screening a phage library for soluble TCRs, TCR-like molecules, andportions thereof is presented in PCT International Patent ApplicationPublication No. WO 2001/062908.

VI. Adoptive T Cell Therapy Utilizing the Presently Disclosed TCRs,TCR-Like Molecules, and Portions Thereof

In some embodiments, the TCRs, TCR-like molecules, portions thereof ofthe presently disclosed subject matter, and T cells comprising the same,can be employed for use in adoptive T cell therapy. Generally, adoptiveT cell therapy relies on the in vitro expansion of endogenous,cancer-reactive T cells. These T cells can be harvested from cancerpatients, manipulated, and then reintroduced into the same or adifferent patient as a mechanism for generating productive tumorimmunity. Adoptive T cell therapy has had promising early clinicalresults and has been associated with clinical responses.

CD8+ cytotoxic T lymphocytes are the primary effector cells in adoptiveT cell therapy. However, CD4+ T cells might also play an important rolein maintaining CD8+ cytotoxic function and transplantation of tumorreactive CD4+ T cells has been associated with some efficacy inmetastatic melanoma. T cells used in adoptive therapy can be harvestedfrom a variety of sites, including peripheral blood, malignanteffusions, resected lymph nodes, and tumor biopsies. Although T cellsharvested from the peripheral blood are easier to obtain technically,tumor-infiltrating lymphocytes (TILs) obtained from biopsies mightcontain a higher frequency of tumor-reactive cells.

Once harvested, T cells can be expanded either through polyclonalstimulation with activating antibodies or through exposure to specifictumor antigens. This second approach requires the identification ofrelevant targets, however. Given the frequency of antigen loss variantsin current clinical trials, the selection of appropriate targets couldbe challenging, potentially making polyclonal stimulation a moreattractive approach. In some embodiments, a relevant target comprises anantigenic fragment of an IRS2 polypeptide (i.e., SEQ ID NO: 2), a CDC25bpolypeptide (i.e., SEQ ID NO: 11), a desmuslin//synemin polypeptide(i.e., SEQ ID NOs: 17 and 18), and/or a β-catenin polypeptide (i.e., SEQID NO: 28). In some embodiments, the antigenic fragment comprises anamino acid sequence as set forth in SEQ ID NOs: 2, 12, 19, 29, or 30.

Several strategies, including the enforced expression of costimulatoryproteins and telomerase, have been used to extend the life span ofcultured T cells. IL-15 has also been considered as a possible additiveto cultures in order to enhance the production of cytotoxic cells.Engraftment of adoptively transferred T cells appears to be enhanced inlymphodepleted hosts, and strategies to combine pretreatment withlymphodepleting chemotherapy and adoptive T cell transplantation appearto increase treatment efficacy significantly.

Two alternative approaches attempt to circumvent low levels ofendogenous antitumor reactivity in the peripheral blood by directlysupplying T cells with the ability to recognize tumors. T cellsharvested from the peripheral blood can be engineered to express TCRs,TCR-like molecules, and/or portions thereof that have been selected fortumor recognition. This approach has been tested in metastatic melanoma.However, because TCR recognition of an antigen is MHC restricted, eachengineered TCR can typically only be used in patients with the requiredMHC allele. MHC restriction can be bypassed by engineering T cells toexpress novel chimeric fusion proteins that link the antigen-bindingdomain of the B cell receptor with the signaling component of the TCRcomplex. These “T-bodies” can directly bind tumor antigens, leading to Tcell activation, but can be used to target only cell surfaceoverexpressed proteins while in some embodiments TCRs, TCR-likemolecules, and portions thereof recognize peptides derived from proteinsin all cell compartments. In some embodiments, the presently disclosedsubject matter employs a T cell that has been modified to express a TCR,a TCR-like molecule, and/or a portion thereof as defined herein.

VII. Other Methods

VII.A. Methods of Treatment Using Conjugates Comprising TCRs, TCR-LikeMolecules, and/or Portions Thereof

In some embodiments, the conjugates of the TCRs, TCR-like molecules, orportions thereof with active agents are used to treat a condition in asubject in need thereof. In some embodiments, the

Exemplary soluble fusion proteins for use with the presently disclosedsubject matter are in some embodiments fully functional and soluble. Bythe term “fully functional” or similar term is meant that the fusionprotein specifically binds ligand. Assays for detecting such specificbinding include, but are not limited to standard immunoblot techniquessuch as Western blotting. Functional fragments of such soluble TCRs andTCR-like molecules are able to bind antigen with in some embodiments atleast 70% of the affinity of the corresponding full-length TCR orTCR-like molecule, in some embodiments at least about 80% of theaffinity of the corresponding full-length TCR or TCR-like molecule, insome embodiments at least about 90% of the affinity of the correspondingfull-length TCR or TCR-like molecule, in some embodiments at least about95% of the affinity of the corresponding full-length TCR or TCR-likemolecule, and in some embodiments greater than 95% of the affinity ofthe corresponding full-length TCR or TCR-like molecule as determined byWestern blot or Surface Plasma Resonance analysis.

VII.B. Methods of Using the Disclosed TCRs, TCR-Like Molecules, andPortions Thereof as Diagnostic Agents

In some embodiments, the presently disclosed TCRs, TCR-like molecules,and portions thereof can be employed as diagnostic agents. By way ofexample and not limitation, the presently disclosed TCRs, TCR-likemolecules, and portions thereof can be employed in a detection and/ordiagnostic assay such as but not limited to immunohistochemistry tolocalize their cognate phosphopeptides in samples from subjects. Forexample, a tumor biopsy could be contacted with a TCR, TCR-likemolecule, and/or a portion thereof that has been conjugated with adetectable label under conditions sufficient for the presently disclosedTCRs, TCR-like molecules, and portions thereof to bind to its cognatephosphopeptide, and this binding can be detected using standardtechniques. Such an approach can be used, for example, for assayingtumor biopsies to determine whether the cells present in the biopsyexpress a given phosphopeptide and, in some embodiments, to what extentthe phosphopeptide is expressed in the tumor cells. For thosephosphopeptides that are expressed specifically by tumor cells, such anapproach can also be used to assess tumor margins by determining whetheror not the cells at the periphery of a tumor biopsy express or do notexpress a given phosphopeptide.

VII.C. Methods of Modifying the Disclosed TCRs, TCR-Like Molecules, andPortions Thereof to Increase Binding Affinity

Methods of testing a TCR, TCR-like molecule, or a portion thereof of thepresently disclosed subject matter for an ability to recognize a targetand/or a cell and for antigen specificity are known in the art. Forexample, Clay et al., 1999, teaches methods of measuring the release ofcytokines (e.g., interferon-γ (IFNγ), granulocyte/monocyte colonystimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), orinterleukin 2 (IL-2)). In addition, TCR function can be evaluated bymeasurement of cellular cytoxicity, as described, for example, in Zhaoet al., 2005.

Additionally, once a TCR, TCR-like molecule, or portion thereof thatbinds to a peptide of interest has been identified, the amino acidsequence of the same (referred to herein as a “reference sequence”) canbe modified to increase its binding affinity for the peptide ofinterest. In some embodiments, a phage library can be constructed thatincludes a plurality of modified TCRs, TCR-like molecules, or portionsthereof, wherein the modified TCRs, TCR-like molecules, or portionsthereof comprise amino acid sequences that include one or moresubstitutions, deletions, or insertions of the amino acids of thereference sequence. In some embodiments, amino acid sequencemodifications are produced in the CDR regions, optionally just the CDR3region, but in some embodiments also including one or both of the CDR1and CDR2 regions. An exemplary method for screening a phage library ofmodified TCRs, TCR-like molecules, or portions thereof is presented inPCT International Patent Application Publication No. WO 2001/062908.

EXAMPLES

The following Examples provide further illustrative embodiments. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following EXAMPLES areintended to be exemplary only and that numerous changes, modifications,and alterations can be employed without departing from the scope of thepresently disclosed subject matter.

Materials and Methods for the EXAMPLES

Cell Line Care.

Breast cancer cell lines were maintained in D-MEM media containing 10%fetal bovine serum (FBS), 2 mM 1-glutamine, 15 mM Hepes, and Pen/Strep(complete). Melanoma, ovarian carcinoma, and colorectal cancer lineswere maintained in complete RPMI (CELLGRO®, Mediatech, Inc. A CorningSubsidiary, Manassas, Va., United States of America; see Zarling et al.,2006). Transfectants of the B lymphoblastoid cell line C1R expressingeither HLA-A2 (C1R-A2) or a chimeric MHC class I molecule consisting ofα1 and α2 domains of HLA-A2 and α3 domain of H-2D^(d) (C1R-AAD) weremaintained in complete RPMI with 300 μg/ml Hygromycin B (CELLGRO®) orG418 (CELLGRO®), respectively (see Zarling et al., 2006).

Human CD8 T-Cell Culture and IFN-γ ELISpot.

Magnetic bead-enriched (Miltenyi Biotec Inc., Auburn, Calif., UnitedStates of America; Catalogue No. 130-096-495) human CD8 T-cells wereco-cultured with irradiated, peptide-pulsed matured DC for 7 days inindividual 96-well microcultures at a 15:1 T-cell:DC ratio (see Tsai etal., 1998). For experiments evaluating memory responses, enriched CD8T-cells were further magnetic bead-enriched for CD45RO⁺ cells (MiltenyiBiotec Inc., Auburn, Calif., United States of America; Catalogue No.130-046-001). An indirect ELISpot was performed as described inSlingluff et al., 2009 using 25,000 cells/well with or without 75,000peptide-pulsed (10 μg/ml) T2 targets. All human protocols were approvedby the Institutional Review Board for Health Sciences Research of theUniversity of Virginia, Charlottesville, Va., United States of America.

Generation of Murine Phosphopeptide-Specific T-Cells.

Murine CD8 T-cells specific for the pIRS-2₁₀₉₇₋₁₁₀₅ (RVApSPTSGV; SEQ IDNO: 2), pCDC25b₃₈₋₄₆ (GLLGpSPVRA; SEQ ID NO: 12), desmuslin (RTFpSPTYGL;SEQ ID NO: 19), and pβ-catenin₃₀₋₃₉ (YLDpSGIHSGV; SEQ ID NO: 29 andYLDpSGIHSGA; SEQ ID NO: 30) phosphopeptides were generated in AADtransgenic mice as described (see Zarling et al., 2000; Zarling et al.,2006). Yellow Fever NS4B₂₁₄₋₂₂₂ (LLWNGPMAV; SEQ ID NO: 54), and M1₅₈₋₆₆Flu (GILGFVFTL; SEQ ID NO: 55) peptides were used as controls. Peptideswere synthesized by GenScript USA Inc. (Piscataway, N.J. United Statesof America) or Bio-Synthesis Inc. (Lewisville, Tex., United States ofAmerica). All protocols were approved by the Institutional Animal Careand Use Committee (IACUC) of the University of Virginia,Charlottesville, Va., United States of America.

Cloning of Phosphopeptide-Specific Murine TCR α and β Chains.

pIRS-2₁₀₉₇₋₁₁₀₅-specific, pCDC25b₃₈₋₄₆-specific,pDesmuslin₄₂₆₋₄₃₅-specific, and pβ-catenin₃₀₋₃₉-specific murine CD8T-cell lines were magnetically enriched for CD8a (Miltenyi Biotec Inc.,Auburn, Calif., United States of America; Catalogue No. 130-049-401) andtotal RNA isolated using PURELINK™ MICRO-TO-MIDI™ Total RNA isolationkit (INVITROGEN™ Corporation, Carlsbad, Calif., United States ofAmerica). cDNA was synthesized from total RNA (3 μg) using theGENERACER™ Kit (INVITROGEN™ Corporation, Carlsbad, Calif., United Statesof America) as described (see Santomasso et al., 2007). 5′-RACE PCR wasperformed using the GENERACER™ 5′ primer and one of three 3′gene-specific primers:

TCR-CαRev (5′-ACTGGACCACAGCCTCAGCGTCAT-3′; SEQ ID NO: 39); TCR-Cβ1Rev(5′-TGAATTCTTTCTTTTGACCATAGCCAT-3′;  SEQ ID NO: 40);  or TCR-Cβ2Rev(5′-GGAATTTTTTTTCTTGACCATGGCCAT-3′;  SEQ ID NO: 41).

RACE PCR products of correct size (˜900 bp) were cloned into thepCR®4-TOPO® vector (INVITROGEN™ Corporation, Carlsbad, Calif., UnitedStates of America). TCR sequences were confirmed in both directions andmatched to the IMGT database available on the World Wide Web atwww<<dot>>imgt<<dot>>org.

Electroporation of IVT RNA Encoding Phosphopeptide-Specific TCR Chains.

IVT RNA of the TCR 43 chains and transfection of OKT3-activated humanCD8 T-cells were performed as described in Johnson et al., 2006 and Zhaoet al., 2005. The 5′ primers included sequences for T7 RNA polymerasebinding and transcription, followed by a Kozak sequence, a start codonand the next 16-17 bp of Vα or Vβ region for each TCR gene while the 3′primers included 66 T residues (T₆₆) and 16-25 bp of the relevant α or βconstant region sequence.

A first pIRS-2₁₀₉₇₋₁₁₀₅-specific TCR α chain cDNA (SEQ ID NO: 3) wasamplified using the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGCTCCTGGCACTCCTCCC-3′; SEQ ID NO: 42), and the 3′ primer (5′-(T₆₆)AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO: 43). A firstpIRS-2₁₀₉₇₋₁₁₀₅-specific TCR β chain cDNA (SEQ ID NO: 5) was amplifiedusing the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGGGCACCAGGCTTCTTGG-3′; SEQ ID NO: 44) andthe 3′ primer (5′-(T₆₆)A GGAATTTTTTTTCTTGACCATGGCC-3′; SEQ ID NO: 45). Asecond pIRS-2₁₀₉₇₋₁₁₀₅-specific TCR α chain cDNA (SEQ ID NO: 7) wasamplified using the 5′ primer(5′-TAATACGACTCACTATAGGGAGAGCCACCATGCTCCTGGCACTC CTCCC-3′; SEQ ID NO:42), and the 3′ primer (5′-(T₆₆)AA CTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO:43). A second pIRS-2₁₀₉₇₋₁₁₀₅-specific TCR β chain cDNA (SEQ ID NO: 9)was amplified using the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGGGCTCCAGACTCTTCTTT-3′; SEQ ID NO: 48) andthe 3′ primer (5′-(T₆₆)AGGAATTTTTTTTCTTGACCATGGCC-3′; SEQ ID NO: 45).

A pCDC25b₃₈₋₄₆-specific TCR α chain cDNA (SEQ ID NO: 13) was amplifiedusing the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGAAGACAGTGACTGGACC-3′; SEQ ID NO: 46) and the 3′ primer (5′-(T₆₆) AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO: 43). A pCDC25b₃₈₋₄₆-specific TCR βchain cDNA (SEQ ID NO: 15) was amplified using the 5′ primer(5′-TAATACGACTCACTATAGGGAGAGCCACCATGTCT AACACTGCCTTCCCT-3′; SEQ ID NO:47) and the 3′ primer (5′-(T₆₆)A GGAATTTTTTTTCTTGACCATGGCC SEQ ID NO:45).

A first pDesmuslin₄₂₆₋₄₃₅-specific TCR α chain cDNA (SEQ ID NO: 20) wasamplified using the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGCTCCTGGCACTCCTCCC-3′; SEQ ID NO: 42), and the 3′ primer(5′-(T₆₆)AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO: 43). A firstpDesmuslin₄₂₆₋₄₃₅-specific TCR β chain cDNA (SEQ ID NO: 22) wasamplified using the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGTCTAACACTGTCCTCGCT-3′; SEQ ID NO: 49) and the 3′ primer (5′-(T₆₆)CTATGAATTCTTTCTTTTGACCATAGCCATCAC-3′; SEQ ID NO: 50). A secondpDesmuslin₄₂₆₋₄₃₅-specific TCR α chain cDNA (SEQ ID NO: 24) wasamplified using the 5′ primer(5′-TAATACGACTCACTATAGGGAGAGCCACCATGCTCCTGG CACTCCTCCC-3′; SEQ ID NO:42), and the 3′ primer (5′-(T₆₆) AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO:43). A second pDesmuslin₄₂₆₋₄₃₅-specific TCR β chain cDNA (SEQ ID NO:26) was amplified using the 5′ primer(5′-TAATACGACTCACTATAGGGAGAGCCACCATGGGCTCC AGACTCTTCTTT-3′; SEQ ID NO:48) and the 3′ primer (5′-(T₆₆)A GGAATTTTTTTTCTTGACCATGGCC-3′; SEQ IDNO: 45).

A first pβcatenin₃₀₋₃₉-specific TCR α chain cDNA (SEQ ID NO: 31) wasamplified using the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGAAGAGGCTGCTGTGTTCT-3′; SEQ ID NO: 51), and the 3′ primer (5′-(T₆₆)AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO: 43). A firstpβcatenin₃₀₋₃₉-specific TCR β chain cDNA (SEQ ID NO: 33) was amplifiedusing the 5′ primer (5′-TAATA CGACTCACTATAGGGAGAGCCACCATGAGCTGCAGGCTTCTC-3′; SEQ ID NO: 52) and the 3′ primer (5′-(T₆₆)AGGAATTTTTTTTCTTGACCATGGCC-3′; SEQ ID NO: 45). A secondpβcatenin₃₀₋₃₉-specific TCR α chain cDNA (SEQ ID NO: 35) was amplifiedusing the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGAACAGATTCCTGGGAATATC-3′; SEQ ID NO: 53), and the 3′ primer (5′-(T₆₆)AACTGGACCACAGCCTCAGCGTC-3′; SEQ ID NO: 43). A secondpβcatenin₃₀₋₃₉-specific TCR β chain cDNA (SEQ ID NO: 37) was amplifiedusing the 5′ primer (5′-TAATACGACTCACTATAGGGAGAGCCACCATGAGCTGCAGGCTTCTC-3′; SEQ ID NO: 52) and the 3′primer (5′-(T₆₆)AGGAATTTTTTTT CTTGACCATGGCC-3′; SEQ ID NO: 45).

Prior to electroporation, donor T-cells were washed three times inserum-free OPTI-MEM® media (Life Technologies Corporation, Gaithersburg,Md., United States of America) and were suspended at 25×10⁶/ml. Cellswere mixed with 2 μg IVT RNA of each TCR α and β chain per 10⁶ cells andtransferred to pre-chilled BTX 2 mm gap cuvettes. Using the BTX T820electroporation system (BTX Instrument Division, Harvard Apparatus, Inc.Holliston, Mass., United States of America), cells were pulsed at 500Vfor 0.3 msec. Transfected cells were placed in AIM-V® brand serum-freemedium (Life Technologies Corporation, Gaithersburg, Md., United Statesof America) with 5% AB⁺ serum (GEMCELL™; Gemini Bio-Products, WestSacramento, Calif., United States of America) for 8-24 hours andevaluated for TCR expression and functionality.

Functional Analysis of Phosphopeptide-Specific Murine TCR-ExpressingHuman CD8 T-Cells.

14-16 hours post-electroporation, phosphopeptide-specific murineTCR-transfected human CD8 T-cells were co-cultured in AIM-V (LifeTechnologies Corporation, Gaithersburg, Md., United States of America)supplemented with 5% human AB⁺ serum (GEMCELL™; Gemini Bio-Products,West Sacramento, Calif., United States of America) with peptide-pulsedor unpulsed C1R-AAD, C1R-A2, or cancer cells endogenously expressing thepIRS-2₁₀₉₇₋₁₁₀₅ or the pCDC25b₃₈₋₄₆ phosphopeptide. Cell surfaceexpression of mouse TCRβ, human CD3, and human CD8 molecules on humanCD8 T-cells were assessed using antibodies from either Becton DickinsonBioscience (San Jose, Calif., United States of America) or eBioScienceInc. (San Diego, Calif., United States of America). During a 5 hourco-culture of stimulator cells with phosphopeptide-specific murineTCR-transfected human CD8-T cells at 37° C., anti-human CD107a-Alexa 647antibody (eBioScience Inc., San Diego, Calif., United States of America)was added in the presence of 5 μg/ml Brefeldin A (SIGMA-ALDRICH® Co.LLC, St. Louis, Mo., United States of America), 5 μg/ml Monensin(eBioScience Inc., San Diego, Calif., United States of America) and 300IU/ml human IL-2 (Chiron Corporation, Emeryville, Calif., United Statesof America). Cells were then stained for surface molecule expression,fixed and permeabilized using CYTOFIX/CYTOPERM™ (Becton DickinsonBioscience (San Jose, Calif., United States of America) and stained forintracellular cytokine (anti-IFN-γand anti-TNF-α, eBioScience Inc., SanDiego, Calif., United States of America). Immunofluoresence was analyzedusing the Becton Dickinson FACSCANTO™ I or FACSCANTO™II flow cytometerand analyzed using FlowJo software (Tree Star, Inc., Ashland, Oreg.,United States of America).

In Vitro Cytotoxicity Assay.

Phosphopeptide-specific murine TCR-expressing human CD8 T-cells wereco-cultured for 5 hours with a 1:1 mix of C1R-A2 cells pulsed with 1 μMphosphopeptide and stained with 1 μM carboxyfluorescein succinimidylester (CFSE; Life Technologies) and unpulsed C1R-A2 cells stained with0.1 μM CFSE. Specific killing was assessed by evaluating percent loss ofthe peptide-pulsed population relative to the unpulsed population.

Western Analysis.

Lysates were generated as described in Zarling et al., 2006 or using theTHERMO SCIENTIFIC™ NE-PER™ protein extraction kit (Thermo FisherScientific Inc., Waltham, Mass., United States of America). Protein wasloaded and separated on 8-16% gradient gel (ISC BioExpress, Kaysville,Utah, United States of America, or THERMO SCIENTIFIC™, Thermo FisherScientific Inc., Waltham, Mass., United States of America) by SDS/PAGE.Lysate created in HEK293T cells (NBL1-08995; Novus Biologicals, LLC,Littleton, Colo., United States of America) was used as a positivecontrol for CDC25b (loaded 1 μg of protein in order to not over-exposeblot). Proteins were transferred to Immobilon FL PVDF (EMD MilliporeCorporation, Billerica, Mass., United States of America) and membranesblocked and probed with pSer¹¹⁰⁰-IRS-specific Ab and GAPDH-specific Ab(Santa Cruz, SC-25778) as previously described (Zarling et al., 2006).The blots were then stripped with Restore Plus (Thermo Scientific) andreprobed with anti-IRS-2 specific antibodies (Santa Cruz, H-205). ForCDC25b, total CDC25b protein was detected with CDC25b Antibody (C-20,Santa Cruz) after first blocking with 5% clarified milk with 0.1% Tween®20 brand nonionic detergent. Pixel density for the staining ofpSer¹¹⁰⁰-IRS-2 or total CDC25b was determined using ALPHAEASEFC™software.

Immunohistochemistry.

Formalin-fixed paraffin-embedded cell line pellets and tissuemicroarrays of metastatic melanoma samples (Biorepository and TissueResearch Facility of the University of Virginia, Charlottesville, Va.,United States of America) were deparaffinized, rehydrated,counterstained with hematoxylin, incubated with anti-Ser¹¹⁰⁰-pIRS-2 for3 hours at 4° C. after antigen retrieval, and specific antibody stainingwas detected using IMMPACT™ AEC (3-amino-9-ethylcarbazole; VectorLaboratories, Inc., Burlingame, Calif., United States of America).Antibodies were removed with ethanol and acidified potassiumpermanganate and then reprobed with anti-IRS-2 (Santa CruzBiotechnology, Inc., Dallas, Tex., United States of America). Acomparison of mean specific staining densities/unit area was performedfor each metastatic melanoma and the adjacent uninvolved tissue usingthe Aperio “Positive Pixel count” (PPC) algorithm on an Aperio Scanner.To calculate specific staining, the PPC of representative sections frompeptide-blocked slides was subtracted from the PPC of correspondingrepresentative sections stained with the anti-Ser¹¹⁰⁰-pIRS-2 antibodywithout blocking peptide.

Tumor Control.

Seven to 8 week old male NOD/SCID/IL-ORγc^(−/−) mice (The JacksonLaboratory, Bar Harbor, Me., United States of America) were inoculatedsubcutaneously with 1.4×10⁶ AAD⁺ SLM2 melanoma cells. 3×10⁶ human CD8T-cells expressing either pIRS-2- or pCDC25b-specific TCR, or 1.5×10⁶ ofboth populations, were adoptively transferred 3 days later. Anadditional 1.5×10⁶ T-cells were given 4 days later. All mice received1500 CU of IL-2 (R&D Systems, Inc., Minneapolis, Minn., United States ofAmerica) imp. every other day for 10 days. Tumor size was measured every2-3 days with a digital caliper, and calculated as L×W (mm²). Tumor freesurvival is equal to the measurement day when the tumor size was >30mm².

Statistical Analysis.

Log-rank (Mantel Cox Test) analysis, Cox proportional hazard modeling,and parametric modeling were performed to determine statisticalsignificance where indicated. p values less than 0.05 were consideredsignificant.

Example 1 Immunogenicity of Phosphopeptides for Human Donors In Vitro

The pIRS-2₁₀₉₇₋₁₁₀₅ and pCDC25b₃₈₋₄₆ phosphopeptides were initiallyidentified on two melanomas and an ovarian carcinoma (Zarling et al.,2006), but their abilities to induce T-cell responses in humans was notevaluated. Thus, T-cells from normal human donors were cultured inreplicate microwells with autologous mature dendritic cells (DC) pulsedwith either pIRS-2₁₀₉₇₋₁₁₀₅ or pCDC25b₃₈₋₄₆. After 7 days, T-cells inthese cultures produced IFN-□ when restimulated withphosphopeptide-pulsed HLA-A2⁺ targets (see FIGS. 1A and 1B; “p” refersto the phosphorylated form). They did not recognize targets pulsed withthe unphosphorylated (IRS-2₁₀₉₇₋₁₁₀₅ or CDC25b₃₈₋₄₆) homologous peptide(see FIG. 1B). The magnitude of these responses was surprisingly high.Donor 44's phosphopeptide-specific responses were significantly greaterthan that to a yellow fever virus peptide (LLWNGPMAV; SEQ ID NO: 54), towhich this donor had not been previously exposed. Donor 54 had beenimmunized with yellow fever vaccine and this individual's phosphopeptidespecific responses were somewhat lower than the yellow fever responsealthough still strong (see FIG. 1A).

It has recently been established that immunity to someleukemia-associated phosphopeptides in normal individuals resides in thecentral memory compartment (Cobbold et al., 2013). Thus, CD45RO⁺ CD8T-cells were isolated from four (4) different donors using magneticbeads and stimulated them with autologous DC pulsed with eitherpIRS-2₁₀₉₇₋₁₁₀₅ or pCDC25b₃₈₋₄₆ for 7 days. Using a cutoff of >50spots/25,000 cells, all four donors showed moderate to strongpre-existing memory responses to the pCDC25b₃₈₋₄₆ peptide, and 2/4donors responded to pIRS-2₁₀₉₇₋₁₁₀₅ (see FIG. 1C). In all cases, theT-cells were specific to the phosphorylated peptide and did notrecognize the unphosphorylated homolog. The magnitude of these memoryresponses was quite variable among peptides and donors, but was in somecases equivalent to or greater than memory responses to influenza oryellow fever epitopes (Note: donors 54 and 62 had been immunized with ayellow fever vaccine and had a strong yellow fever peptide-specificmemory T cell response. Donors 43 and 44 were yellow fever naïve). Thiswas inconsistent with the development of self-tolerance to thesephosphopeptides. Combined, the strength of the responses in FIG. 1 wasconsistent with the possibility that these three normal human donors hadbeen previously exposed to both phosphopeptides. However, none of thesedonors had indications of autoimmune disease, consistent with thepossibility that these phosphopeptides were not displayed on normaltissue.

Example 2 Functional Activity of Phosphopeptide-Specific Murine TCR UponExpression in Human CD8 T-Cells

Recent reports have shown that adoptive transfer of human T-cellstransfected with cloned high affinity tumor-reactive TCR can lead topositive clinical responses in cancer patients (Cohen et al., 2006;Morgan et al., 2006; Johnson et al., 2009; Park et al., 2011). Thesehigh avidity TCR also enable the expression of endogenously processedand presented TAA on cancers of multiple types to be determined.

In order to bypass potential limitations in self-tolerance and thegeneration of unintended cross-reactivities, HLA transgenic mice wereemployed to elicit phosphopeptide-specific murine T-cells from which TCRchains were cloned. AAD mice, expressing a class I MHC molecule thatcontains the α1 and α2 domains from HLA-A2, and the α3, transmembrane,and cytoplasmic domains from H-2D^(d), were immunized with autologous DCpulsed with either pIRS-2₁₀₉₇₋₁₁₀₅ or pCDC25b₃₈₋₄₆. CD8 T-cell linesderived from these animals secreted IFN-γ when cultured with AAD⁺targets pulsed with the phosphorylated forms of these epitopes but nottheir non-phosphorylated counterparts (see FIG. 1D; non-phosphorylatedhomolog indicated by triangles). However, they failed to recognizephosphopeptide-pulsed targets expressing fully human HLA-A2, most likelydue to the low affinity of murine CD8 for the human α3 domain (see Cohenet al., 2006; Jorritsma et al., 2007).

cDNAs encoding the TCR α and β chains from pIRS-2₁₀₉₇₋₁₁₀₅-specific (SEQID NOs: 3 and 5 and 4 and 6, respectively) or pCDC25b₃₈₋₄₆-specific (SEQID NOs: 13 and 15, respectively) T-cell lines were molecularly clonedand utilized as templates to produce in vitro transcribed (IVT) RNA(Zhao et al., 2006; Santomasso et al., 2007). Electroporation of IVT RNAinto either TCR-deficient SupT1 cells or human CD8 and CD4 T-cellsresulted in surface expression as detected by staining for mouse TCRP(FIGS. 2A and 2B). TCR expression was detected at high levels at 9 hours(FIG. 2B) with some TCR still detectable out to 5 dayspost-electroporation (FIG. 2C).

Human CD8 T-cells electroporated with IVT RNA encoding either TCRproduced IFN-γ and/or upregulated CD107a, a marker of cytotoxicactivity, in a dose-dependent manner after co-culture withphosphopeptide-pulsed AAD⁺ targets (see FIGS. 3A and 3B). Both TCRconferred half-maximal recognition at a peptide dose of ˜400-800 pM. Incontrast to the murine T-cells expressing these TCR (FIG. 1D), the humanCD8 T-cells recognized phosphopeptide-pulsed targets expressing HLA-A2at least as well as those expressing AAD (FIGS. 3A and 3B). Neither cellproduced IFN-γ or upregulated CD107a in response to HLA-A2⁺ targetspulsed with high levels of the non-phosphorylated peptide. Human CD8T-cells expressing the pIRS-2-specific murine TCR also killedpIRS-2₁₀₉₇₋₁₁₀₅-pulsed, but not pCDC25b₃₈₋₄₆-pulsed, targets in vitro(FIG. 3A), while those expressing the pCDC25b-specific TCR killedpCDC25b₃₈₋₄₆-pulsed but not pIRS-2₁₀₉₇₋₁₁₀₅ or pβ-catenin₃₀₋₃₉-pulsedtargets (FIG. 3B). Thus, the expression of these murine TCR in human CD8T-cells imparted phosphopeptide-specific, high-avidity recognition andboth cytotoxic and cytokine-secreting effector activities.

Example 3 pIRS-2₁₀₉₇₋₁₁₀₅ and pCDC25b₃₈₋₄₆ Phosphopeptides are BroadlyExpressed on Cancer Cells

Whether these transfected human CD8 T-cells could recognize endogenouslyprocessed and presented pIRS-2₁₀₉₇₋₁₁₀₅ or pCDC25b₃₈₋₄₆ phosphopeptideon HLA-A2⁺ cancer cell lines was investigated. To correlatepIRS-2₁₀₉₇₋₁₁₀₅-specific T-cell recognition with phosphopeptideexpression, an antibody specific for the Ser¹¹⁰⁰-phosphorylated IRS-2protein (pSer¹¹⁰⁰-IRS-2) as well as an antibody that recognizes totalIRS-2 protein (Zarling et al., 2006) were employed. A substantialfraction of pIRS-2₁₀₉₇₋₁₁₀₅-specific T-cells upregulated CD107a, and asubset of these also produced IFN-γ upon co-culture with two HLA-A2⁺melanoma cell lines, MelSwift and 1102Mel (FIG. 3C). These two celllines also expressed high levels of pSer¹¹⁰⁰-IRS-2 (FIG. 4). However, norecognition was evident upon co-culture with an HLA-A2^(neg)pSer¹¹⁰⁰-IRS-2⁺ melanoma, SK-Mel-28, or an HLA-A2⁺, low to negativepSer¹¹⁰⁰-IRS-2 ovarian carcinoma, OV-90. There is no specific antibodyfor Ser⁴²-phosphorylated CDC25b. However, human CD8 T-cells transfectedto express the pCDC25b₃₈₋₄₆-specific TCR recognized two HLA-A2⁺melanomas that expressed high levels of total CDC25b (MelSwift and1102Mel), and failed to recognize either an HLA-A2^(neg) CDC25b⁺melanoma, SK-Mel-28, or an HLA-A2⁺ CDC25^(lo) ovarian carcinoma, OV-90(FIGS. 3C and 5).

These T-cells were then employed to evaluate expression ofpIRS-2₄₀₉₇₋₁₁₀₅ and pCDC25b₃₈₋₄₆ on HLA-A2⁺ cancer cell lines ofdifferent histological origins. For the HLA-A2⁺ cancer cells, Westernblots were loaded based on cell equivalents so it would be possible tocorrelate Ser¹¹⁰⁰-phosphorylated IRS-2 directly with T-cell recognition.Although the amount varied, Ser¹¹⁰⁰-phosphorylated IRS-2 was detected byWestern blot in the majority of melanoma, ovarian cancer, colorectaladenocarcinoma, breast cancer, bladder cancer, and non-small cell lungcancer (NSCLC) lines evaluated, but was poorly expressed in prostatecancer cells (see FIGS. 4A and 4C). None of the bladder, prostate, orNSCLC cancer cells were HLA-A2⁺ and their recognition bypIRS-2₁₀₉₇₋₁₁₀₅-specific T-cells could not be tested.

However, of the HLA-A2⁺ cell lines evaluated, pIRS-2₁₀₉₇₋₁₁₀₅ waspresented by 10/10 melanomas evaluated, 3/4 ovarian carcinomas, 2/2colorectal carcinomas, and 2/3 breast carcinomas (see FIG. 4B). Cancercells that were better recognized by pIRS-2₁₀₉₇₋₁₁₀₅-specific T-cellsalso expressed higher amounts of pSer¹¹⁰⁰-IRS-2 detected by Western blot(FIG. 4D). pCDC25b₃₈₋₄₆-specific T-cells also did not recognize theHLA-A2^(neg) cancer cells T47D and SK-Mel-28 (FIGS. 3C and 5B). They didrecognize 3/4 HLA-A2⁺ melanomas, 3/3 breast cancer lines (FIGS. 3C and5B), and the HLA-A2⁺ EBV-transformed lymphoblastoid cell line JY.

However, although pCDC25b-specific T-cells showed high avidity andhigh-level recognition of peptide-pulsed targets (see FIG. 3B), theirrecognition of these cancer cells was relatively low (see FIG. 5B). Theyalso did not recognize the two colorectal adenocarcinomas and fourovarian cancer cell lines evaluated.

Good pCDC25b₃₈₋₄₆-specific T-cell recognition was associated with highlevel expression of the CDC25b source protein in some cells but lowlevel expression in others, with no correlation between level of sourceprotein and pCDC25b₃₈₋₄₆-specific T-cell recognition (FIG. 5C). Thissuggested that there were differences in the level or the turnover ofpSer⁴²-CDC25b in relation to the total CDC25b protein in differentcancer cells. In sum, pIRS-2₁₀₉₇₋₁₁₀₅ phosphopeptide was endogenouslyprocessed and presented in a large number of cancers of differenthistological origin, and this display elicited strong effector responsesfrom pIRS-2-specific TCR-expressing human CD8 T-cells. In contrast,while pCDC25b₃₈₋₄₆ phosphopeptide was presented by melanoma, breastcancer, and EBV-transformed lymphoblastoid cell lines, its overallexpression was more limited.

Example 4 Mitotically Active Melanoma Cells Express High Levels ofpSer¹¹⁰⁰-IRS-2 Protein

The expression of pSer¹¹⁰⁰-IRS-2 in human melanoma explants and normaltissues was evaluated. Sections from cell blocks containing the positivepSer₁₁₀₀-IRS-2 SLM2 melanoma, the low to negative pSer-IRS-2 OV-90ovarian carcinoma, and a melanoma metastasis to the lung, each of whichhad been stained in the presence or absence of blocking pIRS-2₄₀₉₇₋₁₁₀₅phosphopeptide, were compared. The results are presented in FIG. 6.

As shown therein, addition of the blocking peptide largely eliminatedstaining for all samples. Strong cytoplasmic staining for pSer¹¹⁰⁰-IRS-2was evident in the SLM2 melanoma, with the highest staining in cellswith condensed chromosomes and undergoing mitosis (FIG. 6A). Staining ofmitotic cells was also evident in the OV-90 ovarian carcinoma, but thesecells were a significantly lower fraction of the total cell number, andstaining of non-mitotic cells was very weak (FIG. 6B). This wasconsistent with the very weak pSer¹¹⁰⁰-IRS-2 Western blot staining (FIG.4A) and lack of T-cell recognition by pIRS-2₁₀₉₇₋₁₁₀₅-specific T-cells(FIGS. 3C and 4B). Strong staining was also evident in the humanmelanoma lung metastasis specimen, again with the highest level inmitotic cells (FIG. 6C).

Tissue blocks of metastatic melanoma that included adjacentnon-neoplastic tissue were also evaluated. The non-neoplastic tissuesevaluated were heart (n=1), liver (n=1), lung (n=3), and colon (n=1).Addition of the blocking peptide almost completely inhibited stainingfor all of these samples. The melanoma metastases varied widely in theirlevel of pSer¹¹⁰⁰-IRS-2 (see FIGS. 7A, 7C, and 7E, and Table 2).Increased staining was observed in mitotically active melanoma cells andalso in peritumoral stroma (FIGS. 7A and 8). When quantified as totalstaining intensity per unit area of tissue section, pSer-IRS-2 stainingvaried among normal tissues, but was not convincingly different thanthat in tumors (see Table 2 below). However, there were few mitoticcells and thus few intensely staining cells in most normal tissues(FIGS. 7B, 7D, and 7F). High staining intensities were observed incolonic mucosal epithelial cells adjacent to a melanoma metastasis (FIG.8), and in normal colon biopsies. This might have been indicative of thehigh mitotic activity of colonic epithelial cells. The high staining ofmucosal epithelial cells was mainly limited to those at the epithelialsurface; whereas staining of deeper portions of the mucosa and submucosawas lower than that of the rectal melanoma shown (FIG. 8).

It was also notable that rectal mucosa and stroma immediately adjacentto the rectal melanoma had focally high staining intensity. To explorewhether high staining intensity in normal superficial mucosal epithelialcells in the colon might signal a more global high expression in surfaceepithelium generally, normal skin was also stained. There was lowstaining in the epidermis, much of which persisted despite peptideblocking, indicating that the staining of normal epidermis was largelynon-specific (see FIGS. 7G and 7H).

TABLE 2 Specific Anti-Ser1100-pIRS-2 Staining Density (Positive PixelCount × 10⁷/μ²) for Melanoma Metastases and Surrounding TissuesPseudostratified Respiratory Adjacent Epithelium Tissue Melanoma Normalwithin Peritumoral specimen Metastasis Tissue Normal Tissue Stroma Heartmuscle 2.2 0.4 ND ND with melanoma metastasis Liver with 1.5 4.3 ND NDmelanoma metastasis Lung with 6.1 1.7 5.5 2.8 melanoma metastasis A Lungwith 7.1 9.8 21 14 melanoma metastasis B Lung with 9.4 2.5 ND NDmelanoma metastasis C Colon with 3.3 1.6-34 melanoma metastasis Normalskin A ND  0.42 Normal skin B ND 2.4 Mean in 27 ND ND ND mitotic cells(lung with melanoma metastasis B)** Mean in 419.5 mitotic cells (SLM2melanoma)* Specific staining with anti-Ser1100-pIRS-2 was determined bysubtracting the positive pixel count (PPC) for representative sectionsfrom peptide-blocked slides from the PPC from correspondingrepresentative sections without blocking peptide. ND = not done. **meanfor mitotic cells (n = 5) in Lung B melanoma metastasis. *mean formitotic cells (n = 5) in SLM2 melanoma cells in vitro.

Overall, these data suggested that successful immune targeting ofpSer¹¹⁰⁰-IRS-2: 1) might selectively target dividing malignant cells;and 2) also might target peritumoral stroma. Each of these could supporttumor control. The data also raised the possibility that immunetargeting of pSer¹¹⁰⁰-IRS-2 could carry some risk of adverse effects oncolonic epithelium but not other normal tissues evaluated.

Example 5 Phosphopeptide-Specific TCR-Expressing T-Cells can Slow TumorOutgrowth

Whether these two phosphopeptides could serve as immunotherapeutictargets for treatment of cancer was also tested. NOD/SCID/IL-2Rγc^(−/−)mice were inoculated subcutaneously with SLM2 melanoma cells, and 3 dayslater were injected with human CD8 T-cells expressing either thepIRS-2-specific or pCDC25b-specific TCR, or both populations, togetherwith IL-2. A second infusion of transfected CD8 T-cells was given 4 dayslater. Outgrowth of tumor was evident past day 25 in all groups (seeFIG. 9), most likely due to gradual loss of expression of thephosphopeptide-specific murine TCR (FIG. 2C), or loss of the T-cells.

However, animals that had received phosphopeptide-specificTCR-expressing cells remained tumor-free (tumor size less than 30 mm²)for significantly longer than control animals that only received IL-2(see FIG. 9). Indeed, infusion of either pIRS-2₁₀₉₇₋₁₁₀₅-specific orpCDC25b₃₈₋₄₆-specific CD8 T-cells or both populations resulted indelayed outgrowth in comparison to the control animals. Overall, thisdemonstrated that the endogenous levels of pIRS-2₁₀₉₇₋₁₁₀₅ andpCDC25b₃₈₋₄₆ phosphopeptide on melanoma were sufficient for T-cellrecognition and allowed some delay in tumor growth in vivo.

Discussion of the Examples

Disclosed herein are the characterizations of two new phosphopeptideTAAs, pIRS-2₁₀₉₇₋₁₁₀₅ and pCDC25b₃₈₋₄₆, which are endogenously processedand presented on multiple HLA-A2⁺ cancers. Both phosphopeptides werestrongly immunogenic in vitro for human T-cells and in vivo for HLA-A2transgenic mice, lending credence to their effectiveness as vaccines.Indeed, memory responses in four normal healthy donors to thepCDC25b₃₈₋₄₆ phosphopeptide and 2/4 normal donors to the pIRS-2₁₀₉₇₋₁₁₀₅phosphopeptide were observed. This suggested that these healthyindividuals had been previously exposed to a stimulus that hasestablished immunological memory to these two phosphorylated TAAs.

Immunological responses to cancer-testes antigens is usually only seenin cancer patients, not normal individuals, and is associated with poorprognosis (Scanlan et al., 2002). Tolerance is also believed to limitmuch of the immune response to the tissue-associated differentiationantigens (Colella et al., 2000; Touloukian et al., 2003). Memoryresponses to the pIRS-2₁₀₉₇₋₁₁₀₅ and pCDC25b₃₈₋₄₆ phosphopeptide innormal individuals are evidence of previous encounters with nascenttumors that have disregulated phosphorylation cascades.

To explore the display of endogenously processed and presentedphosphopeptide on cancer cells, the TCRs from murine CD8 T-cell linesspecific for each phosphopeptide were isolated and transfected intohuman CD8 T-cells. A phosphosite-specific antibody was also employed todetermine whether patient tumor samples and cancers of differenthistological origins expressed the phosphorylated IRS-2 source protein.pIRS-2₄₀₉₇₋₁₁₀₅-specific murine TCR-modified human CD8 T-cellsrecognized endogenously processed and presented phosphopeptide onmultiple HLA-A2⁺ melanomas and breast, ovarian, and colorectalcarcinomas, and this recognition correlated with the level of expressionof Ser₁₁₀₀-phosphorylated IRS-2 source protein. Mitotically active cellsalso had the strongest staining for Ser¹¹⁰⁰-phosphorylated IRS-2protein, in both tumors and surrounding peritumoral areas.

pCDC25b₃₈₋₄₆-specific TCR-modified human CD8 T-cells recognizedendogenously processed and presented phosphopeptide on several HLA-A2⁺melanoma, breast cancer, and lymphoblastoid cell lines. Thus, disclosedherein are new reagents that can be utilized to evaluate and treatcancer patients: murine TCR chains specific for either pIRS-2₁₀₉₇₋₁₁₀₅-or pCDC25b₃₈₋₄₆-peptides that can be utilized as immunotherapeuticagents to target patient's immune responses against thesepost-translationally modified epitopes. In addition, thepIRS-2₁₀₉₇₋₁₁₀₅-specific TCR therapy can be combined with Ser¹¹⁰⁰-IRS-2phospho-specific antibody to determine whether patient tumor samplesexpress pSer¹¹⁰⁰-IRS-2 protein.

One approach currently showing some promise for the treatment of cancerpatients involves the adoptive transfer of tumor-specific CD8 T-cells,generated through vaccination and/or by genetic modification viaexpression of TCR chains specific for an appropriate TAA (Rosenberg,2008). Most of the TCR chains currently cloned and studied in humanclinical trials for melanoma have been specific for melanocytedifferentiation proteins (Rosenberg et al., 2004; Rosenberg, 2008; Parket al., 2011). Although of importance for melanoma, extending this formof immunotherapy to antigens that are broadly expressed on other typesof cancers, such as disclosed herein for the pIRS-2₁₀₉₇₋₁₁₀₅ andpCDC25b₃₈₋₄₆ phosphopeptides, can facilitate the broadening of adoptivecell therapy to multiple cancer patients.

In sum, pIRS-2₁₀₉₇₋₁₁₀₅ phosphopeptide was presented by a large numberof cancers of different histological origin, and this display elicitedstrong effector responses from pIRS-2-specific TCR-expressing human CD8T-cells. Similarly, while pCDC25b₃₈₋₄₆ phosphopeptide was presented bymelanoma, breast cancer, and EBV-transformed lymphoblastoid cell lines,its overall expression was more limited.

REFERENCES

All references listed in the instant disclosure, including but notlimited to all patents, patent applications and publications thereof,scientific journal articles, and database entries (including but notlimited to GENBANK® database entries and including all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, and/or teach methodology, techniques, and/orcompositions employed herein. The discussion of the references isintended merely to summarize the assertions made by their authors. Noadmission is made that any reference (or a portion of any reference) isrelevant prior art. Applicants reserve the right to challenge theaccuracy and pertinence of any cited reference.

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It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. An isolated T cell receptor (TCR), or portionthereof, that specifically binds to a phosphopeptide-HLA-A2 complex,wherein the phosphopeptide has the amino acid sequence set forth in SEQID NO: 12, and the isolated TCR, or portion thereof, comprises: an alphachain variable domain comprising a CDR1 region sequence comprising aminoacids 48-53 of SEQ ID NO: 14, a CDR2 region sequence comprising aminoacids 71-77 of SEQ ID NO: 14, and a CDR3 region sequence comprisingamino acids 112-121 of SEQ ID NO: 14; and a beta chain variable domaincomprising a CDR1 region sequence comprising amino acids 56-60 of SEQ IDNO: 16, a CDR2 region sequence comprising amino acids 78-83 of SEQ IDNO: 16, and a CDR3 region sequence comprising amino acids 121-129 of SEQID NO:
 16. 2. The isolated TCR, or portion thereof, of claim 1, whereinthe alpha chain variable domain comprises an amino acid sequence atleast 95% identical to amino acids 22-132 of SEQ ID NO:
 14. 3. Theisolated TCR, or portion thereof, of claim 1, wherein the beta chainvariable domain comprises an amino acid sequence at least 95% identicalto amino acids 30-138 of SEQ ID NO:
 16. 4. The isolated TCR, or portionthereof, of claim 1, wherein the alpha chain variable domain comprisesan amino acid sequence at least 95% identical to amino acids 22-132 ofSEQ ID NO: 14, and wherein the beta chain variable domain comprises anamino acid sequence at least 95% identical to amino acids 30-138 of SEQID NO:
 16. 5. The isolated TCR, or portion thereof, of claim 1, whereinthe alpha chain variable domain comprises the amino acid sequence ofamino acids 22-132 of SEQ ID NO: 14, and wherein the beta chain variabledomain comprises the amino acid sequence of amino acids 30-138 of SEQ IDNO:
 16. 6. An isolated TCR alpha chain, or portion thereof, comprisingan alpha chain variable domain comprising a CDR1 region sequencecomprising amino acids 48-53 of SEQ ID NO: 14, a CDR2 region sequencecomprising amino acids 71-77 of SEQ ID NO: 14, and a CDR3 regionsequence comprising amino acids 112-121 of SEQ ID NO:
 14. 7. Theisolated TCR alpha chain, or portion thereof, of claim 6, wherein thealpha chain variable domain comprises an amino acid sequence at least95% identical to amino acids 22-132 of SEQ ID NO:
 14. 8. The isolatedTCR alpha chain, or portion thereof, of claim 6, wherein the alpha chainvariable domain comprises the amino acid sequence of amino acids 22-132of SEQ ID NO:
 14. 9. An isolated TCR beta chain, or portion thereof,comprising a beta chain variable domain comprising a CDR1 regionsequence comprising amino acids 56-60 of SEQ ID NO: 16, a CDR2 regionsequence comprising amino acids 78-83 of SEQ ID NO: 16, and a CDR3region sequence comprising amino acids 121-129 of SEQ ID NO:
 16. 10. Theisolated TCR beta chain, or portion thereof, of claim 9, wherein thebeta chain variable domain comprises an amino acid sequence at least 95%identical to amino acids 30-138 of SEQ ID NO:
 16. 11. The isolated TCRbeta chain, or portion thereof, of claim 9, wherein the beta chainvariable domain comprises the amino acid sequence of amino acids 30-138of SEQ ID NO:
 16. 12. The isolated TCR, or portion thereof, of claim 1,wherein the isolated TCR, or portion thereof, is conjugated to an activeagent.
 13. The isolated TCR, or portion thereof, of claim 12, whereinthe active agent is a detectable label selected from the groupconsisting of biotin, streptavidin, an enzyme or catalytically activefragment thereof, a radionuclide, a nanoparticle, a paramagnetic metalion, and a fluorescent, phosphorescent, or chemiluminescent molecule.14. The isolated TCR, or portion thereof, of claim 12, wherein theactive agent is an immunostimulatory molecule.
 15. The isolated TCR, orportion thereof, of claim 14, wherein the immunostimulatory molecule isa CD3 agonist wherein said CD3 agonist is an anti-CD3 antibody.
 16. Theisolated TCR, or portion thereof, of claim 12, wherein the active agentis a therapeutic agent selected from the group consisting of analkylating agent, an antimetabolite, a natural product havingpharmacological activity, a mitotic inhibitor, an antibiotic, acytotoxic agent, and a chemotherapeutic agent.
 17. The isolated TCR, orportion thereof, of claim 1, wherein the isolated TCR, or portionthereof, is humanized, comprises a human constant domain, or both.
 18. Apharmaceutical composition comprising the isolated TCR, or portionthereof, of claim
 1. 19. The pharmaceutical composition of claim 18,wherein the pharmaceutical composition further comprises an adjuvantselected from the group consisting of montanide ISA-51, QS-21, tetanushelper peptides, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG),corynbacterium parvum, levamisole, azimezone, isoprinisone,dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freund'sadjuvant (complete and incomplete), mineral gels, aluminum hydroxide(Alum), lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, dinitrophenol, diphtheria toxin (DT).
 20. A kit comprising:(i) a cytokine and/or an adjuvant; and (ii) a composition comprising theisolated TCR, or portion thereof, of claim 1.